Data from the HIV-Alcohol Longitudinal Cohort was used to determine the prevalence of substance use by victims and assailants during physical and sexual assault against HIV-infected persons and whether these findings differed by gender. Of the sexually assaulted participants, 31% of victims and 70% of assailant(s) had used drugs/alcohol during sexual assault. Compared with men, women had higher odds of substance use during sexual assault (adjusted odds ratio [OR] 3.8, 95% confidence interval [CI] 1.6 to 8.7) and of substance use by their assailant(s) during sexual assault (adjusted OR 5.9, 95% CI 1.7 to 20.6) in adjusted analysis. Of the physically assaulted participants, 66% of victims and 85% of assailants used drugs/alcohol during physical assault; these results did not differ by gender.