Berkhamsted lost 2-1 at Homer Green on Tuesday in the South Midlands Premier Division.
Berko got off to a fine start taking the lead after six minutes when James Baldry headed in a corner.
But the lead lasted just eight minutes until Joe Collins levelled the contest. The winning goal came through Greg Deer's strike and the hosts held on despite late pressure.
Sixth-placed Berkhamsted crushed Oxhey Jets 8-2 on Saturday with Alex Campana netting four times before the break.
Campana scored twice in the space of a minute to put the hosts in control. But Luke Lloyd's shot deflected off Dale Sears to give the visitors hope of a comeback.
Lloyd conceded a penalty which Campana converted for his hat-trick and the goalscorer netted again with a confident finish.
Ade Kilani scored a spectacular overhead kick for the Jets. But this was wiped out by a goal from Adiel Mannion.
Jordan Gilbert netted twice and a second from Mannion ensured the hosts hit Oxhey for eight.
Berko are at home to EBAC Northern League Division One side Morpeth Town tomorrow in the fifth round of the FA Vase.
The club are hoping to secure their biggest crowd since they were formed in 2009.
Admission for the cup tie is £6 for adults and £3 for seniors with free entry for accompanied young children.
Sun Sports were beaten 3-0 at Hoddesdon Town on Saturday.
Comments: Our rules
We want our comments to be a lively and valuable part of our community - a place where readers can debate and engage with the most important local issues. The ability to comment on our stories is a privilege, not a right, however, and that privilege may be withdrawn if it is abused or misused.
Please report any comments that break our rules.
Read the rules here