Background In the past few years, there has been an expanding global interest in the problem of substance use. A variety of studies conducted within the past decade have investigated the prevalence of substance use among university students. The study aimed to detect the prevalence and associates of substance use among Egyptian students at Mansoura University. This cross-sectional, questionnaire-based, observational study was conducted during the period from February to July 2019 on 1138 university students at Mansoura University in Egypt. The university students were randomly divided into three classes (medical, practical, and theoretical). The data were assembled using questionnaires of sociodemographic, clinical attributes, and drug use disorders identification test (DUDIT). Results Lifetime substance use among students was 6.5%. Of the users, 18 (24.3%) used cannabis, 14 (18.9%) used tramadol, 10 (13.5%) used alcohol, 5 (6.8%) used benzodiazepines, and 1 (1.4%) used heroin. Only 35.1% used polysubstance, of those, 10 (13.5%), 12 (16.2%), 3 (4.1%), and 1 (1.4%) used cannabis and tramadol; cannabis and alcohol; cannabis and inhalant; and cannabis, alcohol, and tramadol respectively. There was a substantial association between substance use and male sex, age above 20 years, smoking, and living in urban areas. In the group that used substances, 50 (4.4%) suffered substance-related problems and 6 (0.5%) encountered dependency. Conclusion In total, using the mono substance is more than using poly substances in which cannabis, tramadol, and alcohol were the most often utilized substances. The highest percentage of drug-related problems was among medical students while dependence was highest among practical students. These results should be considered in future substance preventive programs.