Southend strengthened their hopes of automatic promotion from League Two while weakening Oxford's chances of a play-off place.
The U's started well and Dean Morgan forced Cameron Belford into a smart save, with Jon-Paul Pittman lively.
But Ryan Hall fired the Shrimpers ahead with a 25-yard free-kick that flew past debutant keeper Connor Ripley.
Bilel Mohsni doubled the lead when he headed Peter Gilbert's cross into the bottom corner.
Southend are in fifth place, but lie just a point behind Crawley in third place heading into the final weekend.
Oxford, meanwhile, are in eighth place, three points behind in-form Crewe.
VIEWS FROM THE DRESSING ROOM
Southend United manager Paul Sturrock told BBC Essex:
"The fat lady still hasn't sung yet, so we go into the last game of the season with something to play for."
"I'm delighted with the performance. The work-rate and attitude of the players was unbelievable.
"Right through the second half, it was going to back to the walls, but the boys worked very hard. They put in a shift.
"Defensively we were very sound today another clean sheet, which we're delighted about."
Oxford United 31 Ripley 02 Batt 05 Duberry Booked 06 Wright Booked 16 Whing (Montano - 75' ) 08 Heslop (Chapman - 46' ) 14 Hall 26 Davis 11 Pittman 18 Morgan (Craddock - 57' ) 23 Rendell Substitutes 34 Crocombe 17 Capaldi 07 Chapman 27 Montano 29 Craddock Southend United 35 Belford 02 Clohessy 03 Gilbert 06 Bilel 12 Ferdinand 15 Phillips Booked 23 Barker 04 Hall 07 Grant 08 Timlin 09 Harris Substitutes 16 Prosser 34 Hills 14 Martin 24 Benyon 37 Eastwood