Marcus Rashford scored the only goal of the game with a penalty after Jack Grealish was fouled in the area.
The Three Lions had another chance to score from the spot but Jordan Henderson, who came off the bench, saw his effort saved.
Southgate made nine changes to his side after England beat Austria at the Riverside on Wednesday, and will now have to decide who takes the final place in his 26-man squad following an injury to Trent Alexander-Arnold.
Former Sunderland player Jordan Henderson came off the bench at half-time as he continues to step up his recovery from injury.
England have a week to prepare ahead of their Euro 2020 opener against Croatia at Wembley on Sunday, June 13.
Here’s how Southgate’s men fared against Romania.
. England's midfielder Jordan Henderson gestures during the international friendly football match between England and Romania at the Riverside Stadium in Middlesbrough, north-east England on June 6, 2021. - - NOT FOR MARKETING OR ADVERTISING USE / RESTRICTED TO EDITORIAL USE (Photo by SCOTT HEPPELL / POOL / AFP) / NOT FOR MARKETING OR ADVERTISING USE / RESTRICTED TO EDITORIAL USE (Photo by SCOTT HEPPELL/POOL/AFP via Getty Images)
England's midfielder Jordan Henderson. Photo: SCOTT HEPPELL
. Sam Johnstone - 7
Made an excellent save in the second half when on the floor. A few routine stops as he kept his clean sheet intact. 7 Photo: Stu Forster
. Ben Godfrey - 6
Played at right-back and wasn’t asked too many questions defensively in the first half. Caught out a few times but stuck to his task. 6 Photo: Stu Forster
. Ben White - 7
Looked comfortable on the ball and made a few important interceptions. England’s defence didn’t look totally convincing though. 7 Photo: Stu Forster
. Tyrone Mings - 6
A few nervy moments as Romania caught England on the counter attack a couple of times and the defender was out of position. 6 Photo: Getty Images
1. Sam Johnstone - 7
Made an excellent save in the second half when on the floor. A few routine stops as he kept his clean sheet intact. 7 Photo: Stu Forster
2. Ben Godfrey - 6
Played at right-back and wasn’t asked too many questions defensively in the first half. Caught out a few times but stuck to his task. 6 Photo: Stu Forster
3. Ben White - 7
Looked comfortable on the ball and made a few important interceptions. England’s defence didn’t look totally convincing though. 7 Photo: Stu Forster
4. Tyrone Mings - 6
A few nervy moments as Romania caught England on the counter attack a couple of times and the defender was out of position. 6 Photo: Getty Images