Fiorente's win in front of 100,000 racegoers at Flemington on Tuesday, sealed Waterhouse's standing as one of Australia's greatest horse trainers.
It also gave Damien Oliver his third Melbourne Cup, two months after he returned to racing from serving a ban for betting on a rival horse in a race he was riding in in 2010.
Fiorente staged a thrilling duel with Red Cadeaux before gaining the upperhand at the 100m. Mount Athos finished third.
Waterhouse's victory came after twice training the runner-up, Te Akau Nick in 1993 and Fiorente in last year's race.
It also confirmed Waterhouse as one of the most resilient figures in Australian racing.
She has been in the headlines since a public fall-out with high profile owner John Singleton and an ongoing battle with Sydney racing stewards.
Waterhouse trained most of Singleton's racehorses until the glamour mare More Joyous was beaten in the All Aged Stakes during the Sydney autumn racing carnival.
Singleton claimed Waterhouse's son, the bookmaker Tom Waterhouse, had told rugby league star Andrew Johns that More Joyous couldn't win the All Aged before the mare ran unplaced in the race.
Oliver, who won the race on Doriemus and Media Puzzle, brought Fiorente from midfield to stake his challenge at the 300m.
He said it was a privilege to win the race for Waterhouse.
"It's amazing, it's a dream come true," he said.
"I'm so rapt that I could be part of Gai's first one. She's done so much for racing and it's a great honour for me to help bring home her first one.
"Gai was one of the first people to get behind me when I came back and I can't thank her enough for helping me get going again and we have been a great team together."
Fiorente, who ran second to Green Moon in last year's race, started the $7 favourite with Red Cadeaux at $61 and Mount Athos going out at $13.
Irish stayer Simenon made a long run in the race and fought on bravely for fourth with Dandino taking fifth.
Caulfield Cup winner Fawkner was the first Australian-bred horse across the line, finishing sixth.
Waterhouse paid Oliver the ultimate compliment for his ride.
"He rode him a treat," she said.
"He got back to about 12th and it was quite a solidly run race and then he just kept tracking up and tracking up. He was just fantastic, it's a dream come true."
TRAGEDY MARS CUP FOR FRENCH
Two years after the high of a Melbourne Cup win, French jockey Christophe Lemaire hit rock bottom in a tragic end to his dream of a second Cup.
Lemaire won on Dunaden in 2011 and went into Tuesday's renewal confident he could repeat the result on the Aga Khan's mare Verema.
But the horse fractured a cannon bone at the 2000m mark and could not be saved.
"It's just bad luck," Lemaire said.
"There were no signs before the race something like that would happen.
"She was on the bridle travelling well alongside the future winner, we were in a good position, but these kinds of things just happen."
While Lemaire was "very sad", Verema's strapper was inconsolable after the Cup.
"She was a good mare. I won a couple of Group races with her in France," Lemaire said.
"You come here dreaming about winning the race, not having this happen halfway round."
SBS reporter Rhiannon Elston caught some of the colour and excitement ahead of the race, and the focus was on the fashion.
Jockeys, trainers and owners of the 24 Melbourne Cup starters weigh in on their chances:
1: DUNADEN - Mikel Delzangles (Trainer): "I think he's in very good form, the same as he was two years ago, but I think he is even stronger. The thing is he has four kilogram more, so he needs to be."
2: GREEN MOON - Nick Williams (Part owner): "He's obviously got to improve, he's got a bit more weight this year but it would be a great story if he could go back-to-back. It would be something very special to us."
3: RED CADEAUX - Ed Dunlop (Trainer): "He loves it here and he's in very good form. We could have done with a better draw though."
4: SEA MOON - Nick Williams: "He's in good order and has really come on since his last run in the Herbert Power. I think you'll see him at the absolute top of his game."
5: BROWN PANTHER - Michael Owen (Owner): "My horse has travelled well. He only lost about seven kilos on the flight and he's put that back on. He's eating fine, he's training well. We've got all the boxes ticked so far but obviously the biggest box to tick is to be good enough to win the race."
6: FIORENTE - Damien Oliver (Jockey): "He's had a great preparation and is ready to go. It's a great opportunity; hopefully I can make the most of it."
7: FORETELLER - Chris Waller (Trainer): "He is a seven-year-old, he's not going to get another chance and it's an amazing race. He's probably got the best form lines going into it, if he can stay."
8: DANDINO - Darren Dance (Part owner): "We can't have the horse any better. It looks like we're going to get a firm track. We've got a good gate. A good rider. It's the hardest Melbourne Cup field, probably the most even. There's 13 Group One winners. We're not a Group One winner yet, but let's hope we are after Tuesday."
9: ETHIOPIA - Pat Carey (Trainer): I was delighted with Ethiopia's run in the Lexus Stakes. The winner Ruscello ran a cracker to win and we were pleased with how our lad was finished.
10: FAWKNER - Nick Williams: "Fawkner has certainly gone forward since the Caulfield Cup and I think he'll run a great race."
11: MOURAYAN - Nick Williams: "I thought he was average (in the Mackinnon Stakes) but as he has got older he's been inclined to put those flat runs in before he runs a good two-mile race.
12: SEVILLE - Nick Williams: "He's fantastic. I think he's got a really good chance."
13: SUPER COOL - Mark Kavanagh (Trainer): "It was a good run in the Cox Plate and I'm sure we'll have him right for Melbourne Cup."
14: MASKED MARVEL - Nick Williams: "The thing with him is, he's the best stayer in this field. If he runs up to and finds his English form, he'll win. But the key to him is that he's got a few tricks and we haven't had him that long."
15: MOUNT ATHOS - Marwan Koukash (Owner): "We will bloody win."
16: ROYAL EMPIRE - Saeed Bin Suroor (Trainer). "We thought this is the right horse. He has done everything right so far. I'm happy with his condition, physically and mentally he is doing really well. There's no excuse."
17: VOLEUSE DE COEURS - Mike Moroney (Trainer): "She hasn't put a foot wrong since being here. She's been a dream to deal with and has done everything we've asked her to do. She ticks all the boxes."
18: HAWKSPUR - Jim Cassidy (Jockey): "He's hard fit and I think he's really ready to run the race of his life. I wouldn't be getting off mine to ride any other one."
19: SIMENON - Willie Mullins (Trainer): "I'm really happy how he's taken his training, how he's taken his quarantine and how he's travelled."
20: IBICENCO - Simon O'Donnell (co-owner) : "The Geelong Cup is one of the two best form races for the Cup. He is a genuine two miler and has always been a genuine contributor."
21: VEREMA - Christophe Lemaire (Jockey): "I'm very confident with her. She's tough and very mature for this kind of race. She likes fast ground so she's got a lot of positive points."
22: DEAR DEMI - Clarry Conners (Trainer): "She couldn't be any better. She's happy, I'm happy and Singo's happy."
23: TRES BLUE - Tommy Berry (Jockey): "He's shown me all the indications that he'll run a strong two miles and he's got good tactical speed so that helps as well."
24: RUSCELLO - Ed Walker (Trainer): "We're enjoying the ride. I just hope the horse can perform well. It's a huge ask. We're under no illusions but he's an in-form improving horse."