Background Nantes University Hospital comprises 20 activity sectors.Aims To investigate the role of the work environment at the individual level, as well as the workplace level, in explaining the variability in employees’ perception of stress. Methods A self-administered enhanced Karasek Job Content Questionnaire was sent to employees. The main variables were the psychological job demand (PJD) score and the job decision latitude (JDL) score. Univariate and multivariate logistic regression analyses were conducted to estimate crude odds ratio (OR) and adjusted OR. Results One thousand eight hundred and sixty-eight workers were included. Nursing managers (25.9 ± 3.4), non-specialized nurses (25.6 ± 3.5) and physicians (25.3 ± 3.4) had the highest PJD. Cleaning staff (61.4 ± 11.4) and nurse aides (63.6 ± 8.8) had the lowest JDL. Items correlated with high PJD are: unacceptable work schedule, adjusted OR 2.16 (95% CI = 1.3–3.5); unsatisfactory workstation accessibility, OR 1.92 (95% CI = 1.1–3.2); getting from A to B, OR 1.67 (95% CI = 1.2–2.4); and heavy manual handling, OR 1.62 (95% CI = 1.1–2.3). Sleeping tablet use was linked to high PJD (P < 0.01), extra workload (P < 0.05) and tiredness (P < 0.05). Use of painkillers was correlated with musculoskeletal disorders (P < 0.05). Conclusions Our study highlighted women >40 years old, nurse managers, physicians, permanent and/or full-time workers having a high PJD. Nursing aides, medical secretary and nurses presented with high strain. Better control measures should be implemented for those socioprofessional categories to improve prevention measures. This study should be repeated in the future with a multi-centre approach to determine the generalizability of the findings.