In their first loss since the season's opening day, Spurs took the lead but were overhauled at Cheshunt FC as Brighton ran out 4-1 winners.
They still have a chance to progress in the competition but it looks likely they will now need a positive result at WSL 1 table-toppers Chelsea in their final group game scheduled for December 6.
Chelsea also top the group after two wins in two with Brighton and Spurs also on six points having played three games. Brighton take on Bristol City in their final match.
Spurs are back at Cheshunt FC on Sunday as they take on Doncaster Belles in WSL 2. Kick-off is 2pm.
Below: Hannah Mackenzie and Asheligh Neville go up for a header against Brighton (photo: Wu's Photography)
Tottenham Hotspur Ladies’ perfect start to Group Two South in the FAWSL Continental Tyres Cup was halted after Brighton & Hove Albion turned around a 1-0 deficit to record a 4-1 victory at Theobalds Lane.
Sarah Wiltshire and Josie Green had early efforts before Spurs found the initiative on 15 minutes. A move starting with Renee Hector from the back culminated in Coral-Jade Haines' perfectly-weighted pass sending Bianca Baptiste through on the inside right and she calmly slotted across Lucy Gillett for 1-0.
However, Brighton equalised three minutes later when Sophie Perry’s curled ball got away from Toni-Anne Wayne for Lucy Somes to score from close. Renee Hector cleared off the line on 21 minutes.
Baptiste's attempted chip was stopped by Gillet and the same player fired a free-kick over.
The Seagulls took the lead with 31 minutes played. Perry’s diagonal free-kick dropped to the far-post where Fern Whelan scrambled home.
Josie Green and Baptiste went close as Spurs finished the half on the front foot.
Into the second half and Sarah Wiltshire was next to test Gillet, the goalkeeper alert to tip a dipping effort around the post. Wendy Martin was inches away from Lucia Leon’s cross on 78 minutes.
Ini Umotong secured the win for Brighton with two goals in the final five minutes.
Report from Khalid Karimullah at Cheshunt FC, Theobalds Lane
Photographs with thanks to Wu's Photography
Spurs Ladies: Toni-Anne Wayne, Sophie Mclean (Lucia Leon 57), Renee Hector, Jenna Schillaci, Ashleigh Neville, Hannah Mackenzie (Ronnell Humes 80), Maya Vio, Josie Green (Wendy Martin 65), Coral-Jade Haines, Sarah Wiltshire, Bianca Baptiste. Substitutes (not used): Chloe Morgan, Ryah Vyse, Katie O’Leary, Lauren Pickett.
Brighton & Hove Albion: Lucy Gillett, Beth Roe, Laura Rafferty (Vicky Ashton-Jones 88), Fern Whelan, Sophie Perry, Kirsty Barton, Danni Buet (Natalie Taylor 90), Jenna Legg, Fliss Gibbons, Aileen Whelan (Ini Umotong 76), Lucy Somes. Substitutes (not used): Chloe Sansom, Amelia Ritchie, Bronwen Thomas.
Spurs take on Doncaster Belles in WSL 2 at Cheshunt FC on Sunday, kick-off 2pm.