Spurs, from the FA Women's Premier League South, pushed FAWPL National Division team Watford all the way in their latest fixture in Group Five.
Unfortunately, two goals in the final eight minutes saw Watford home 2-0 and they now have to beat Colchester by three clear goals in the final to have any chance of progressing to the knockout stages.
"The girls can be proud of their performance and we'll have plenty of confidence for the match against West Ham on Sunday," said coach Juan Amoros.
It's back to FAWPL South action against the Hammers at Thurrock FC in Grays, Essex on Sunday, kick-off 2pm.
SPURS LADIES 0-2 WATFORD
The ladies had the best of the first half as Wendy Martin and Chloe Burr went close. Bianca Baptiste was a constant threat but defences remained on top and it was 0-0 at half-time.
Siobhan Wilson replaced Chloe at half-time and had the best chance of the game after Bianca broke forward on the counter-attack, but a defender slid in to block in front of goal.
Jo Ayre also saved brilliantly but Watford took the lead in the 82nd minute with a header from a corner.
The team pushed on again but Watford secured their win with another goal from a set-piece.
Juan added: "The team's work on pressuring Watford was fantastic and we held them until the 82nd minute, when a set-play decided the game.
"We still have a small chance in the competition. We will travel to Colchester to try to get the three points and wait to see the other results. For us, to be behind Watford and Barnet (National Divison teams) but on top of West Ham and Colchester (in their Southern Division) would be a good result."
Spurs Ladies: Ayre, McEvoy, Pittuck, Cheeseman, Schillaci, Maloney (Downie, 83), Cooper, Smith (O'Halloran), Burr (Wilson, 46), Baptiste, Martin.
For more information on Spurs Ladies visit tottenhamhotspur.com/spurs-ladies