Objectives: This study aimed to estimate the prevalence of psychoactive substance (PAS) use in night-shift healthcare workers (NSHW) during France's first COVID-19 wave (March-May 2020). Design: Observational cross-sectional online survey. Setting: 39 public hospitals in the Assitance Publique des Hôpitaux de Paris (AP-HP) network in the Parisian area. Participants: A total of 1238 nurses, assistant nurses, X-ray technicians, managers, lab technicians, midwives and childcare assistants working at night or alternating between days and nights answered the questionnaire. Intervention: Online survey. Outcome measures: PAS use prevalence after weighting data for sex, age and profession using calibration on margins, in order to be representative of all AP-HP NSHW. We used the Fagerström scale and the Alcohol Use Disorders Identification Test Concise to assess PAS use. Results: The weighted estimated prevalences of daily smoking, alcohol drinking and tranquilliser use in participating NSHW were 21.4, 1.3 and 2.4%, respectively. Twelve per cent (11.7%) of our study sample used opioids. During the first COVID-19 wave, PAS use remained stable except for tobacco use, with 8.6% of participants reporting an increase. Previous 3-month prevalences of tranquilliser and opioid use were significantly higher than in the general population. Conclusion: Daily smoking (especially in younger men) and tranquilliser and opioid use were highly prevalent in NSHW in the AP-HP network during France's first COVID-19 wave. Specific interventions for quitting smoking and addressing determinants of tranquilliser and opioid use in NSHW need to be developed and evaluated to improve quality of life in these essential, underdiagnosed and undertreated health personnel.