This paper is the result of a research that evaluated the levels of prejudice against sexual and gender minorities within 28 Brazilian public schools. The research considered a sample of 413 teachers, 97 employees, and 1829 students from 28 public high schools, located in four Brazilian states: Rio Grande do Sul, Minas Gerais, Ceará, and Pernambuco. All of them answered a questionnaire on sociodemographic data, the revised version of the Prejudice Against Sexual and Gender Diversity scale. The resulting analysis highlighted that religious individuals and followers of the Neo-Pentecostal church in the three study groups presented higher levels of prejudice than the other groups involved. All groups that have done previous training in the subject of prejudice presented inferior scores to those that had not done. Individuals that stated they have gay man, lesbian woman, travestis persons, or transsexual persons as friends, relatives, and acquaintances in the groups of teachers and students presented a lower level of prejudice compared to those who did not have relationships with people with these characteristics. Our results suggest the need for methodological changes in schools so that institutions can prepare their curriculum and their pedagogical practices considering the current multiple existing sexual and gender orientations.