Arsenal and Manchester City had already crashed out of the UEFA Champions League on Tuesday and Wednesday respectively, and Spurs will need to come from behind for the second time in as many rounds if they are to make the quarter-finals after a disappointing home defeat.
Rodrigo scored the opener on the half-hour mark before Luisao doubled the advantage after the break – both scoring their second goal in as many matches.
With progression looking an unlikely prospect for Spurs after the second strike, Christian Eriksen pulled a goal back with a 64th-minute free-kick, but Luisao dashed Tottenham hopes in the closing stages when he found the net in the aftermath of a Benfica free-kick.
The result represents Spurs' first home defeat in 12 European fixtures, and Benfica's three away goals leave them with a mountain to climb in next week's second leg.
Tottenham boss Tim Sherwood handed Harry Kane just his second start of the season after openly criticising his side that crumbled to a 4-0 Premier League defeat at Chelsea on Saturday, but both teams struggled to carve out any goalscoring opportunities in the opening stages.
The hosts enjoyed plenty of possession in their opponents' half as the first period progressed, but Benfica goalkeeper Jan Oblak remained unchallenged aside from a Kyle Walker free-kick from which Younes Kaboul was deemed to have impeded the Slovenian goalkeeper.
However, Oblak was almost left red-faced in the 25th minute when he carelessly rushed to the edge of his box to meet a seemingly harmless Kaboul header, but scampered back to jump on the ball after letting it bounce past him.
Despite Spurs' greater share of possession, it was the 2013 Europa League runners-up who drew first blood as Rodrigo latched onto a defence-splitting Ruben Amorim pass before finding the bottom left-hand corner of the goal with a low left-footed effort.
Eriksen – who returned from a back injury – threaded the ball into the path of Emmanuel Adebayor shortly after the break, but the striker dragged his attempt well wide of the left-hand post before Aaron Lennon directed a tame strike straight at Oblak.
Tottenham goalkeeper Hugo Lloris had to be on high alert in the 57th minute as he tipped over an Amorim effort after Kane had sloppily conceded possession, but it was not long before the visitors doubled their advantage.
From the resulting corner, Luisao shook off his marker to head beyond a helpless Lloris.
The two-goal advantage did not last long as Eriksen curled home a superb free-kick shortly afterwards to salvage some hope for the London club.
Benfica looked to have ended Tottenham's chances in the competition in the 84th minute though, as Luisao smashed home from close range after Lloris had kept out Ezequiel Garay's header.
The goal prompted a frank exchange of words between Sherwood and Benfica boss Jorge Jesus but it was the latter who had the last laugh, as his side put one foot in the competition's quarter-finals with a terrific road win.
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