Barnet failed to make it three consecutive friendly wins against higher-league opposition.
Following wins against Watford and Charlton, the Bees found League One Southampton a tough nut to crack.
A Joseph Mills goal just after half-time was enough to give the Saints victory.
Barnet once again included Steve Kabba in their line-up. Kabba, who has been offered a contract, played almost an hour.
A crowd of 851 watched the match.
Barnet return to Underhill on Saturday, when Peterborough United are the visitors. Before then, the Bees travel to Wingate & Finchley this evening (Wednesday).
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 The ability to comment on our stories is a privilege, not a right, however, and that privilege may be withdrawn if it is abused or misused.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