Dons led for 75 minutes against the Cougars despite being down to 12 men but ultimately lost 26-28 at the Keepmoat Stadium.
Defeat saw Richard Horne’s side drop from third to fifth in the final league standings and they now must face in-form Hunslet in an eliminator on Sunday.
Had they finished third or fourth in the table they would have instead played in a qualifier – with the loser awarded the luxury of a second bite of the cherry.
Hunslet rounded off the regular season with a morale-boosting 32-14 win at second-placed Workington Town.
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“It’s a huge week for us,” said Horne. “They’re in really good form, I think they’ve won their last four. They’re travelling really well.
“It’s the tough route now. You have to win every game or you’re out of the competition.
“We know what’s at stake now so it’s down to us to make sure the boys are ready for Sunday.
“It’s a bonus that we’re at home on a pitch that we like and in front of our own fans to help spur us on and hopefully get the result.”
The RFL this week confirmed that Ben Cockayne, sent off against Keighley for what the officials saw as a headbutt, will not face any further punishment.
Doncaster had the led the game 26-12 but the visitors piled on the pressure and went over three times in the final 12 minutes to claim victory.
“The sending off changed the game,” said Horne.
“I couldn’t be prouder of the boys, they worked so hard to stay in the game after the sending off.
“We were into total control of the game up until that point and looked as though we could kick on further at 12-0 and rolling them.
"They got us right at the end. We knew we would be hanging on with the energy we used throughout the game being down to 12 men.
“I’m disappointed for the boys because they worked so hard and probably deserved the win.
"We just didn’t get the calls today and that really hurt us. It’s Hunslet now and we’ll just prepare that.”
Barrow have been promoted as champions. Keighley travel to North Wales in the qualifier.