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Background: Fisherfolk are a most-at-risk population for HIV being prioritized for the scale up of HIV treatment in Uganda. Heavy alcohol use and potential drug use may be a major barrier to treatment adherence for men in this setting. Objective: This study examines the prevalence of substance use, and its influence on antiretroviral treatment (ART) adherence, among male fisherfolk on ART in Wakiso District, Uganda. Methods: This cross-sectional study included structured questionnaires (N = 300) with men attending HIV clinics near Lake Victoria. Using generalized logistic modeling analyses with a binomial distribution and logit link, we conducted multivariate models to test the association between each alcohol variable (quantity and frequency index, hazardous drinking) and missed pills, adjusting for covariates, and tested for interactions between number of pills prescribed and alcohol variables. Results: Thirty-one percent of men reported sub-optimal adherence. Half (46.7%) reported drinking, of which 64.8% met criteria for hazardous drinking. Illicit drug use was low (6%). In the multivariate model, men with greater scores on the alcohol frequency and quantity index were more likely to report missed pills compared to those reporting no drinking (AOR: 1.60, 95% CI: 1.29–1.97). Hazardous drinking had a greater effect on missed ARV doses among men taking twice daily regimens compared to once daily (AOR: 4.91, 95% CI: 1.68–14.37). Conclusions: Our findings highlight the need for targeted alcohol-reduction interventions for male fisherfolk on ART who drink at high quantities to improve ART adherence and to prevent the known negative health effects of alcohol for HIV-infected individuals. [ABSTRACT FROM AUTHOR]