Background: The risk of substance use is increasing among university students, especially medical students. This study aimed to investigate the frequency of smoking, alcohol consumption, and substance use and its relationship with general health indicators among the students of Guilan University of Medical Sciences (GUMS) in 2020. The differences in the frequency of substance use were also investigated from 2005. Methods: This was a cross-sectional analytical study conducted on 406 students of GUMS, Iran, in 2020. The data were collected through three online questionnaires including a demographic questionnaire, the Alcohol, Smoking, and Substance Involvement Screening Test (ASSIST), and the General Health Questionnaire (GHQ). The data were entered into SPSS software version 22 and analyzed using Chi-square, Fisher’s Exact tests, and logistic regression at a significance level of 0.05. Findings: In this study, 53.9% of the participants were female, 46.1% were male, and 54.4% of them experienced using substances in their lifetime. The highest prevalence of use was in consumers of tobacco (46.8%), alcoholic beverages (34.7%), and cannabinoid compounds (19%). Besides, 48.5% of the participants were suspected of having some degree of psychiatric disorders, among which depression (11.6%) and anxiety (8.4%) were the most common. Moreover, substance use during students’ lifetime had a statistically significant association with anxiety, depression, and mental health. Conclusion: The results of this study showed the prevalence of smoking, alcohol consumption, and substance use among the students of GUMS is worrying. The relationship between consumption and consumer’s health indicators highlights the necessity of intervention and purposeful planning by policymakers in this field.