The aim of this work was to evaluate the profile of military police officers regarding the use of psychoactive substances and the presence of Sexually Transmitted Infections (STI). Cross-sectional study carried out with military police officers in 2015 in the central-western, Brazil. The ASSIST (Alcohol, Smoking and Substance Involvement Screening Test) questionnaire was applied to investigate the use of psychoactive substances and serology was performed for syphilis, viral B and C hepatitis and HIV. Cronbach's alpha and Pearson Correlation Coefficient were employed. The ASSIST presented alpha coefficients of Cronbach almost perfect for tobacco (α=0.83) and substantial for the use of alcohol (α=0.70). Of 657 police officers, 78.5% consumed psychoactive substances at some point in their lives, with 76.7% alcohol, 28.5% tobacco, and 5.2% illegal psychoactive substances. A short intervention was required for 23.3% of police officers who used psychoactive substances, and 1.4% should be referred for treatment. Tobacco use was strongly associated with the use of more than one psychoactive substances in life (φ=0.9327), and the use of marijuana showed a moderate correlation with cocaine/crack (φ=0.5241). The prevalence of STI was 14.0%, being 7.6% for HBV, 6.8% syphilis, 0.5% HIV, and 0.3% HCV. HBV/syphilis and HBV/HIV co-infection were observed in 1.1% and 0.1%, respectively. There was no correlation between STI and use of psychoactive substances. The prevalence of HBV and syphilis was higher among police officers than in the general population. The ASSIST questionnaire was consistent when applied to this group and can be a significant tool for monitoring and decision making for timely intervention.