After a weekend's break due to waterlogged pitches, the ladies travelled to Beckenham Town FC to take on the Lionesses, joint-top of the FA Women's Premier League South with six wins in seven at start of play.
Jess Cheeseman was denied by Kylie Davies in the Millwall goal as Spurs started well and then Amy Cooper's fine effort came back off the woodwork.
Andra Marcoci, who has stepped up to the first team after impressing for the reserves, then did well to create space but curled wide.
Millwall took the lead on the half-hour through Emma Whitter and the Lions took a firmer grip on the game in the second half, forcing Ebony Heath and Sam Pittuck to work overtime in central midfield.
The decisive blow arrived five minutes from time when Alicia Ferguson rifled a shot into the top corner.
Manager Karen Hills reported: "We're disappointed because I felt we worked hard but couldn't find the back of the net. We created more chances, which is pleasing, but there was no end product."
The ladies travel to Colchester on Sunday for their final group game in the FA Women's Premier League Cup.
They sit fourth after three games and need to win to have any chance of progressing.
After that, they open the defence of the London Cup with a quarter-final tie at Tower Hamlets.
Spurs Ladies v Millwall: Ayre, O’Halloran, Pittuck, Heath, Schillaci, Maloney (Wilson, 75), Cooper, Baptiste (Burr, 65), Martin, Marcoci, Cheeseman.