Nearly 100,000 people have signed the petition put together by animal rights group PACMA calling for the health minister to quit after nursing assistant Teresa Romero's dog Excalibur was killed.
The petition to be presented in parliament at 12pm on Wednesday also calls for for Madrid health boss Javier Rodríguez and top official Julio Zarco to step down for their role in the animal's death.
The dog was put down earlier in October over concerns it could spread the deadly disease — a move which led to angry confrontations between police and protesters outside Romero's home.
The decision to kill the animal even provoked a global Twitter campaign with people posting pictures of their own dogs alongside messages of support for Excalibur.
"Teresa's dog Excalibur was sacrificed despite the opposition of her owners, thousands of citizens and experts, without evening knowing if it has been infected (with Ebola) and knowing that there are no studies which show that Ebola can be transmitted from dogs to humans," Pacma said in a statement.
For many people in Spain, the death of Excalibur symbolized the authorities' poor reaction to a crisis brought on when nursing assistant Teresa Romero contracted the disease after treating two Spanish missionaries infected with the virus who had been repatriated with the virus.
She has since recovered, although she remains in isolation at Madrid's Carlos III hospital as a precaution.
The crisis saw health professionals calling for Spain's health minister Ana Mato to resign over claims they were poorly trained and inadequately equipped to treat cases of the virus.
Madrid health boss Javier Rodríguez also came under fire after he suggested Romero may have responsible for contracting Ebola by incorrectly following hospital procedures. He also suggested the nursing assistant lied about the state of her health before finally being quarantined — claims fervently denied by her husband Javier Limón.
The Pacma petition will be presented just hours after after the Spanish government voted against an opposition petition calling for her resignation in the wake of Spain's Ebola crisis.The lower house of Spain's party, where the ruling Popular Party has an overwhelming majority, voted against a Socialist-led plan to have Mato ejected from office.
The petition saw most of Spain's opposition groups banding together in a bid to see Mato go over "massive errors" in her management of Spain's Ebola crisis.
The Spanish government rejected the calls, and Mato will remain in office.
The Popular Party did, however, unofficially demoted Mato during Spain's recent Ebola crisis, placing Deputy Prime Minister Soraya Sáenz de Santamaría at the head of an emergency government committee to deal with the control of the virus.