Background The lockdown periods imposed in 2020 by governments had deleterious consequences on population mental health. Several studies based on declarative data have suggested that the lockdown periods were associated with changes in psychoactive substance use but few relied on toxicological analyses. Aims We studied the impact of lockdowns on the pattern of routine care toxicological screening performed on patients hospitalized at the emergency ward (EW) and intensive care units (ICU) at the Grenoble University Hospital. Method This was a retrospective, monocentric study comparing routine care toxicology biological tests performed in children older than 12 years of age and adults hospitalized at the ICU and EW in 2018, 2019, and 2020. Alcohol, illicit drugs, and medications were screened. Generalized linear models were generated to evaluate the effect of the lockdown periods on toxicology results, considering age and sex. Results The study included 13,910 samples from 11,786 patients. There was no significant difference in the repartition of sex or age over the three years. The frequency of positive toxicological tests increased during the lockdown periods (adjusted odds ratio (OR) 95% confidence interval (CI): 1.14, (1.01–1.28), p = .026). The frequency of poly-exposures also rose during these periods (OR 1.43 (1.11–1.82), p = .004) mostly among men (OR 1.54 (1.02–2.04), p = .022), 12–25-year-old patients (OR 1.69 (1.07–2.31), p = .016), and seniors (>56 years) (OR 1.54 (1.00–1.97), p = .032). Conclusions This study suggests that lockdown episodes were associated with increased incidence of psychoactive substance poly-exposures, highlighting the need for preventive strategies for high-risk populations.