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.