Although recent research has highlighted that lesbian, gay, bisexual and transgender (LGBT) youths represent a resilient population, they still suffer from social stigma and oppression, being potentially at additional risk of developing negative mental health outcomes. One of the main environments where violence and harassment against this population are present is the school. Within school contexts, the impact that teachers can have on the educational experiences of LGBT youths seems to be a crucial point. This paper explored sexist, homophobic and transphobic attitudes among 438 pre-service teachers in relation to specific socio-demographic features. Results indicated that being male, heterosexual, conservative and currently religious were positively associated with sexist, homophobic, and transphobic attitudes and feelings, and having a LGBT friend was negatively associated with homophobic and transphobic attitudes and feelings. These results suggest the need to introduce specific training on the deconstruction of gender and sexual stereotypes and prejudices, to provide teachers with efficient tools to address diversity in the classrooms and to implement inclusive school policies. Suggestions for the implementation of good practices are provided.