Jarred Bowen scored the only goal of the game on 59 minutes, finding the top corner from an acute angle with a spectacular effort.
Captain Michael Dawson was then shown a red card with 13 minutes remaining after bringing down his man on the edge of the Tigers’ box but Leonid Slutsky’s men held on to claim an impressive victory.
Kevin Stewart made his first appearance in black and amber following his move from Liverpool on Friday, while Ondrej Mazuch was brought into the starting eleven as a late enforced change for Josh Clackstone.
Benfica’s Horta had the first opportunity of the game after winning the ball in midfield, but his low shot was comfortably held by Allan McGregor.
Abel Hernández was then heavily involved around the half hour mark. First, the Uruguayan appeared to be pulled down inside the area, only for referee Nuno Almeida to wave away his protests. Then the striker let fly with a superb strike from distance but was unlucky to see his effort cannon off the top of the crossbar and away from danger.
Former Fulham striker Mitroglou had the final chance of the first half but McGregor was again down sharply to deny the Greek striker as the teams headed back to the dressing rooms all square.
The Tigers continued to hold their own at the start of the second half and were ahead when Bowen curled home his second spectacular goal in as many games. The winger picked the ball up on the right side of the area and superbly found the top left corner with a stunning effort.
Benfica then began to press Slutsky’s team further up the pitch and McGregor again had to be at his best to deny Jonas’ effort from inside the box.
The game was then spun on its head 13 minutes from time when skipper Dawson saw red for bringing down Seferovic outside the area and the Tigers were left to battle on with ten men.
McGregor continued his fine game between the posts with a string of excellent saves as Benfica pushed for the equaliser and when he was beaten Seferovic could only crash a close range effort against the post as the Tigers held on for victory.
Slutsky’s men will now travel back to Hull tomorrow morning as they begin to prepare for Tuesday’s game against FC Nantes at KCOM Craven Park (KO 7.30pm).
Hull City: Allan McGregor, Brian Lenihan, Michael Dawson, Ondrej Mazuch (Max Clark 65), Ola Aina, Kevin Stewart, Sam Clucas (Dan Batty 83), Jarrod Bowen (Josh Clackstone 78), Markus Henriksen, Kamil Grosicki (James Weir 65), Abel Hernandez.
Subs Not Used: Jon McLaughlin, Greg Olley, Greg Luer.
Benfica: Julio Cesar, Lisandro, Luisão, Samaris, André Horta, Mitroglou, Carrillo, Eliseu, Gonçalves, Carvalho, Buta.
Subs: Fejsa, Filipe Augusto, Jonas, Paulo Lopes, Seferovic, Kalaica, Zivkovic, Cervi, Pereira, Willock, Rada, Arango, Varela, Jardel, Hermes, Chrien, Dias.