The Shrimpers hit back from a goal down to exact revenge on Cheltenham Town for their defeat at the same stage of the competition a year ago.
Cheltenham were forced to withstand some pressure from Southend, who controlled the early proceedings.
Shrimpers striker James Walker tested Scott Brown with two firm shots in quick succession, but the Robins goalkeeper was equal to them.
Loan defender Matt Heath was left all alone in the penalty area to meet Alan McCormack's cross in the 20th minute, but he guided his header against the left post.
Cheltenham came to life as the half wore on and only two excellent pieces of goalkeeping from Steve Mildenhall prevented them taking the lead.
David Bird's pass was dummied by Elvis Hammond, allowing David Hutton to race through on goal, but Mildenhall reacted well to deflect his low shot away from danger in the 38th minute.
Moments later he blocked a powerful header from Michael Townsend, who arrived late to meet Hutton's free-kick delivery from the right.
But Cheltenham opened the scoring during first half stoppage time when Bird's cross fell to Andy Gallinagh, whose attempted shot was forced over the line by Elvis Hammond from close range.
A poor back pass from skipper Shane Duff put Brown under pressure and his poor clearance fell to Barnard, but Brown recovered to smother the shot.
Southend levelled when Heath headed Simon Francis' cross back into the six-yard box and Barnard stooped to head over the line in the 76th minute.
The winner came with two minutes to go when Francis crossed for Barnard to nod home at the far post and send the 176 travelling fans wild.