INTRODUCTION: University professors are highly susceptible to work-related stress, and psychoactive substance use is often used as a stress alleviation strategy. This issue has attracted the attention of organizations that represent these workers given its potential repercussions on work and personal life. OBJECTIVES: This study aimed to evaluate the association between work-related stress and psychoactive substance use in university professors. METHODS: A descriptive and analytical-qualitative study was conducted in a public university in the countryside of the state of Rio Grande do Norte, Brazil. Data were collected from 67 professors using Google Forms and the following instruments: sociodemographic characteristics and occupational activity questionnaire; Alcohol, Smoking and Substance Involvement Screening; and Stress Symptom Inventory. Descriptive methods were used to calculate means and standard deviations. The association between occupational stress, substance use, and the variables studied was investigated using chi-square or Fisher’s exact tests. RESULTS: Most participants were men, married, with children, and a master’s-level education. The mean age of the sample was 42 years. Differences were observed between the prevalence of legal and illegal substance use. Many participants were in the alert, resistance, or exhaustion stages of stress, with the resistance stage being the most frequent. Alcohol was the substance most commonly associated with work-related stress. CONCLUSIONS: Psychoactive substance use is associated with work-related stress among university professors.