Spanish players fumed at the final whistle over the performance of Romanian official Vlad Iordachescu, with players having to be restrained from making physical contact with the referee.
The Spanish Rugby Federation said on Sunday that they had lodged a formal complaint to Rugby Europe and World Rugby.
Spain's defeat meant it was the referee's home nation Romania (and not them) who secured direct passage to the finals in Japan.
Spain wasn't happy with the referee after losing 18-10 to Belgium in a Rugby World Cup qualifying match.
"Following this meeting, a statement will be released to Rugby Europe Board of Directors for dissemination".
Spain must now beat Portugal in a playoff to maintain any hopes of reaching the 2019 tournament in Japan, with the victor facing Samoa in a two-legged tie.
There were ugly scenes after the game which you can see in the video above. Instead, Romania remained one point ahead of Spain despite a 25-16 loss to Georgia on Sunday.
Controversially, Vlad Iordachescu - from Romania, who qualified at Spain's expense - was in charge of the clash and appeared to make a series of contentious decisions in Belgium's favour.More news: NCAA Tournament: Tar Heels routed by Texas A&M
Rugby World Cup 2019 will kick off on September 20, 2019, at Tokyo Stadium.
Rugby Europe responded by saying the appointment of Iordachescu was made independently.
"As for other global major competitions, referees' appointments for Rugby Europe Championship have been made ahead of the competition by an independent and neutral skilled committee".
Spain's coach Santiago Santos was equally furious.
"The difference between penalties against us and for us was huge", Santos said.
"While we appreciate that this is a subject of high-emotion, it would be inappropriate to comment on match official performance before the usual full review has been undertaken".
If they beat Portugal but lose to Samoa they will play in a four-team repechage event in November in a last attempt to qualify for the World Cup for the second time following their appearance in 1999.
Jaime Nava, captain of Spain, said: "I don't want to think that rugby has been prostituted to leave us out".