Neff fought a tough battle to win by one second over Norway's Gunn-Rita Dahle Flesjaa, with road world champion Pauline Ferrand Prevot claiming the third podium spot.
Australia's Rebecca Henderson finished eighth in the women's event, 3min 19sec behind Neff. Australian Road champion Peta Mullens finishing 27th, 7min 50sec down.
In the past four years of UCI World Cup racing in Nove Mesto there have been only two winners of the men's race - Kulhavy took the first edition, and Nino Schurter (Scott-Odlo) the last three. Once again, it came down to these two riders.
Schurter attacked on the start loop, with Kulhavy, World Champion Julien Absalon (BMC), Australian Champion Dan McConnell (Trek Factory Racing) and Florian Vogel (Focus XC) the only riders to stay with him. Schurter surged again on the first full lap, and this time only Kulhavy could hang on.
Schurter appeared to be stronger, gapping Kulhavy on the climbs, but the Czech rider would always come back, possibly motivated by the cheers of thousands of his fans around the course. In the final half lap the tables were turned, when Kulhavy attacked and Schurter could not respond. The Czech rider rolled into the finishing arena to stands full of cheering fans, to win by 24 seconds.
Absalon and Vogel first dropped McConnell, and then Absalon went solo mid-race to take third ahead of Vogel, with Mathias Fluckiger (Stoeckli) taking the fifth and final podium spot. McConnell eventually finished 11th, over four minutes down.
"It was a very exciting race between me and Nino (Schurter) again, and I attacked on the hardest climb and Nino stayed with me maybe 5 or 10 seconds before I got away. Then I just controlled my position to the finish. The finish was good and I didn't make any mistakes. The fans were so amazing. They helped a lot and pushed me. It was crazy. I would like to say thanks to them." - Jaroslav Kulhavy
The UCI Mountain Bike World Cup presented by Shimano continues next weekend in Albstadt, Germany, with Round 2 of the cross-country.
Elite Women: 23.3 km (Start loop + 5 laps)
1 Jolanda Neff (SUI) Stoeckli Pro Team 1hr 34min 29sec
2 Gunn-Rita Dahle Flesjaa (NOR) Multivan Merida Biking Team 0:00:01
3 Pauline Ferrand Prevot (FRA) Raboliv 0:01:39
4 Catharine Pendrel (CAN) Luna Pro Team 0:02:21
5 Annika Langvad (DEN) Specialized Racing 0:02:27
6 Irina Kalentyeva (RUS) Moebel Maerki 0:02:54
7 Lea Davison (USA) Specialized Racing 0:02:55
8 Rebecca Henderson (AUS) Trek Factory Racing 0:03:19
9 Helen Grobert (GER) Ghost Factory Racing 0:03:35
10 Maja Wloszczowska (POL) Kross Racing Team 0:03:36
Elite Men: 27.4 km (1 Start Loop + 6 Laps)
1 Jaroslav Kulhavy (CZE) Specialized Racing 1hr 33min 27sec
2 Nino Schurter (SUI) Scott-Odlo MTB 0:00:24
3 Julien Absalon (FRA) BMC Mountainbike 0:01:16
4 Florian Vogel (SUI) Focus XC Team 0:02:02
5 Mathias Fluckiger (SUI) Stoeckli Pro Team 0:02:37
6 Ralph Naef (SUI) BMC Mountainbike Racing 0:02:59
7 Andrea Tiberi (ITA) FRM Factory Racing 0:03:04
8 Thomas Litscher (SUI) Multivan Merida Biking 0:03:13
9 Manuel Fumic (GER) Cannondale Factory Racing XC 0:03:45
10 Jose Antonio Hermida Ramos (ESP) Multivan Merida Biking 0:03:53
11 Daniel McConnell (AUS) Trek Factory Racing 0:04:17