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Abstract Use of alcohol and other substances remains a major health concern among higher learning institutions. This study aimed at assessing the prevalence of alcohol and other substance use among students at Busitema University in Eastern Uganda. A cross sectional survey was conducted among 658 undergraduate students using a questionnaire consisting of Alcohol, Smoking and Substance Involvement Screening Tool and participant sociodemographic and clinical factors. Logistic regression was used to explore the associations. Two hundred sixty-five (40.3%) students reported ever using alcohol and 158 (24.0%) had used in last 3 months. Seventy-four (11.2%) students reported ever use of other substances including tobacco, cannabis, cocaine, stimulants sedatives and hallucinogens and 36 (5.5%) had used within the recent 3 months. After controlling for potential confounders, recent alcohol use was associated with engaging in romantic relationship (odd ratio (OR) = 1.9, P value (P) = 0.045) while having chronic medical conditions was protective (OR = 0.3, P = 0.031). On the other hand, recent use of other substances was 7 times higher among males (OR = 7.0, P = 0.008) compared to females while fourth year of study was protective (OR = 0.05, P = 0.011). Although alcohol use is a worsening challenge among university students, use of other substances is also highly prevalent after COVID-19 lockdown. There is need for universities to identify students with above factors and design interventions to address them in order to prevent the likely undesirable outcomes of alcohol and substance use.