The Tykes are on the verge of securing a spot in the playoffs, while the Millers are running out of games to avoid relegation.
Daryl Dike's second-half overhead kick proved decisive at the John Smith's Stadium, with the forward scoring his ninth goal in 17 games since signing on loan from Orlando City in January.
That result, combined with Reading's goalless draw against Luton Town on the same evening, put Barnsley six points clear of the seventh-placed Royals with just three matches left to play.
Valerien Ismael's side could all but confirm their place in the top six with victory over the Millers before Reading even face Swansea City on Sunday.
Barnsley's remarkable run of form since the beginning of February, winning 12 and losing just two of their 16 matches, means that they are set to achieve their highest league finish since 2000.
However, one thing they will be wary of is a dip in form at home; the Tykes have won just one of their last four games at Oakwell, dropping points to other lowly teams Derby County and Sheffield Wednesday in recent matches.
Rotherham will aim to follow their fellow strugglers, but they have not beaten Barnsley in their last 10 attempts since 1982, and the Tykes were 2-1 winners in this season's reverse fixture in December.
Now the Millers need a victory more than ever, as their status in the Championship hangs in the balance. Currently in 22nd, they are four points adrift of safety with five games left to play.
Paul Warne's side do have two games in hand over 21st-placed Derby County, who appear in absolute freefall, but they must start picking up points themselves to stand any chance of staying in the second tier.
There was hope that they might do that on Wednesday night as Angus MacDonald gave them the lead after just three minutes against Middlesbrough, but a Matt Crooks red card appeared to change the game and Boro came back to win 2-1.
Therefore it is three defeats on the bounce for Rotherham, who journey to high-flying Brentford on Tuesday after their Oakwell trip.
"We're going to have to lick our wounds, it's going to hurt me for a bit and it's hurt our players," Warne said after the Boro loss. "We have to try and pick them up again tomorrow and attempt another cup final."
The Millers' away record this season is the best in the Championship bottom five, and they have actually conceded fewer goals on the road than Barnsley themselves.
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Dominik Frieser picked up a hamstring injury just before the interval against Huddersfield, so Dike is expected to replace him in the Barnsley starting lineup.
After playing 90 minutes on Wednesday, Carlton Morris may drop to the bench, with Ismael previously mentioning how the club are having to carefully manage the striker's groin problem.
Michael Sollbauer came back into the side against Huddersfield and won man of the match, so should keep his place this weekend.
Rotherham have confirmed that they have appealed Crooks's red card against Middlesbrough but will not know if he is available until Friday.
After four months on the sidelines, Joe Mattock made his return from injury in midweek, but Warne has admitted the left-back will have to be carefully managed and therefore could drop to the bench.
Barnsley possible starting lineup:
Collins; Sollbauer, Helik, Andersen; Brittain, Palmer, Mowatt, Styles; Woodrow, Chaplin; Dyke
Rotherham United possible starting lineup:
Johansson; Ihiekwe, Wood, A. MacDonald; Olosunde, Barlaser, S. MacDonald, Wing, Harding; Smith, Ladapo
We say: Barnsley 1-1 Rotherham United
With Barnsley almost certain of a place in the playoffs, this game arguably means more to Rotherham, and we expect the Millers to get a point at Oakwell; whether that will be enough to help avoid relegation is another matter, but it would at least stop their current losing streak and give them something to build on in the final four games.