Vasco lost the second stage semi-final tie 4-1 on penalties and their exit means Romario will have to wait until the start of the Brazilian championship in mid-May for another chance to reach the milestone.
Romario has failed to hit the target in his last three games and has been stuck on 999 goals for 280 minutes' playing time. The run has also seen Vasco knocked out of the Copa Brasil by second division Gama.
The 41-year-old former Brazil forward was centimetres away from scoring in the 32nd minute when he just failed to make contact with Jorge Luiz's chip as it flew into the goal for Vasco's third.
An extraordinary match produced three goals in the first four minutes and seven in the first half.
Renato and Abedi pounced on defensive mistakes as Vasco went 2-0 ahead in three minutes before Luciano Almeida pulled one back for Botafogo, who equalised with a Ze Roberto goal in the 22nd minute.
Vasco went back ahead 10 minutes later when Jorge Luiz left two defenders in his wake and chipped the ball over goalkeeper Julio Cesar. Romario attempt to get his head to the ball as it crossed the line but just missed.
Dodo and Lucio Flavio put Botafogo 4-3 ahead at halftime but Vasco sent the game to penalties when Alan headed the equaliser in the 37th minute.
In between, an ugly incident delayed the game for four minutes.
Botafogo midfielder Tulio was sent off for insulting the referee and Botafogo team officials ran onto the field to protest. One manhandled the referee before being forced away by police.
Shortly afterwards, Vasco had Andre Dias sent off for a sliding tackle.
There was a 20-minute delay between the end of the match and the penalties as dozens of reporters came onto the field to interview the players.
Botafogo will face either Volta Redonda and Cabofriense in the second stage final. The winners then meet Flamengo, who won the first stage, to decide the championship.
Bragantino completed the line-up for the play-off stage of the Paulista (Sao Paulo state) championship as a 2-1 win over Barueri guaranteed them fourth spot in the qualifying stage.
They joined Santos, Sao Paulo and Sao Caetano in the semi-finals of the championship, which uses a different format to the Carioca tournament.
Bragantino pipped Palmeiras, who won 3-0 at Sao Bento, on goal difference.
Santos completed an impressive campaign by beating Juventus 2-0 to finish top with 50 points out of a possible 57.