The Waders needed to win their re-arranged match at Tamworth last night (Thursday) to keep their hopes alive but they slipped to a 3-1 defeat.
The hosts raced into a 3-0 half-time lead thanks to a Eoin McKeown hat-trick and, although Francis Mampolo replied with a goal just past the hour, Biggleswade could find no way back.
It means the Waders will be playing at Step 4 next season and they are likely to be in the Southern League Division One Central alongside Biggleswade FC.
A message from the club on Twitter following their relegation read: “Thank u to all our volunteers, supporters, Chairman, committee, management and players.
"It’s been a tough season for many reasons. However, we have last game on Saturday but unfortunately after tonight’s game we will be relegated. Time to reflect, regroup + rise again.”
The Waders round of their season with a home clash against Redditch United tomorrow (Saturday).