The bill was passed during the closing session of the legislature on Friday, which brought to an end an 86-day run for the province's politicians.
As was the case with a similar recent law in Ontario, some religious leaders in Manitoba have said Bill 18 violates the religious freedom of faith-based schools by forcing them to accept gay-straight alliances.
More than 300 Manitobans spoke at public hearings on the bill.
Supporters said it will promote tolerance and diversity while opponents said it will do nothing to prevent bullying.
Education Minister Nancy Allan said gay-straight alliances provide a safe place for young people during a vulnerable time in their lives.
The Tories tried to suggest changes to the bill to remove a reference to hurt feelings, saying teachers and coaches could be deemed bullies for simply telling a student they were performing poorly.
MLAs will return to the legislature on Nov. 12.