OBJECTIVE: To develop and validate a questionnaire to assess the knowledge of mechanical ventilation among final-year medical students in Brazil. METHODS: A cross-sectional study conducted between October 2015 and October 2017 involving 554 medical students was carried out to develop a questionnaire for assessing knowledge on mechanical ventilation. Reproducibility was evaluated with the intraclass correlation coefficient, internal consistency was evaluated with Cronbach's alpha, and construct validation was evaluated with a tetrachoric exploratory factor analysis. To compare the means of the competences among the same type of assessment tool, the nonparametric Friedman test was used, and the identification of the differences was obtained with Dunn-Bonferroni tests. RESULTS: The final version of the questionnaire contained 19 questions. The instrument presented a clarity index of 8.94±0.83. The value of the intraclass correlation coefficient was 0.929, and Cronbach's alpha was 0.831. The factor analysis revealed five factors associated with knowledge areas regarding mechanical ventilation. The final score among participants was 24.05%. CONCLUSION: The instrument has a satisfactory clarity index and adequate psychometric properties and can be used to assess the knowledge of mechanical ventilation among final-year medical students in Brazil.