On the hardcourts in Bogota on Tuesday, world No.131 Ebden went down to Japan's Tatsuma Ito 1-6 6-3 6-4.
Millman was comprehensively eliminated by American Alexander Sarkissian 6-3 6-3.
That leaves Bernard Tomic and Sam Groth as the remaining Australians in the tournament.
Tomic is the defending champion and second seed, and after being handed a first-round bye, is due to face the winner between Adrian Menendez-Maceiras and Alejandro Gonzalez.
Groth has made a quick dash to Bogota from Darwin after scoring two pressure Davis Cup wins last Sunday.
Down 2-0 in the quarter-final tie with Kazhakstan, the Victorian partnered Lleyton Hewitt to win the doubles rubber, then replaced Nick Kyrgios for the reverse singles and claimed victory to help lift Australia to a 3-2 triumph.
At a career-high world ranking of No.64, sixth seed Groth will take on Argentinian Guido Pella in Bogota.
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