We previously reported that elevated levels of diazepam binding inhibitor (DBI), also called 'endozepine' because it acts as an endogenous benzodiazepine equivalent on the gamma-aminobutyric acid type A receptor, constitutes a potential risk factor for the diagnosis of non-small cell lung cancer (NSCLC). Antibody-mediated neutralization of DBI improved the immunosurveillance of NSCLC in preclinical models with and without immunotherapy targeting programmed cell death protein 1 (PD-1). A pilot study in a small French-Canadian cohort ( = 205) suggested that benzodiazepine (BZD) use correlates with reduced progression-free survival in NSCLC patients receiving PD-1/PD-L1 blockade. Here, we report a retrospective analysis of the nation-wide French registry of advanced NSCLC patients treated with pembrolizumab. Among the eligible NSCLC patients surviving ≥2 months after treatment initiation ( = 31,479), 37.7% ( = 11,878) received at least two prescriptions of benzodiazepines within 90 days before to 30 days after treatment initiation. Compared to non-users ( = 19,601), BZD users had significantly reduced overall survival (hazard ratio = 1.08, 95% CI: 1.04-1.12,  < 0.001), an effect that persisted after correction using inverse probability of treatment weighting (IPTW) on sociodemographic, clinical, oncologic, and comedication variables. In a subset of 556 patients from the ONCOBIOTICS study, benzodiazepine use was associated with signs of intestinal dysbiosis and alterations in the TOPOSCORE, a prognostic marker linked to poorer outcomes in cancer patients receiving immunotherapy. We conclude that benzodiazepine use may be an independent negative prognostic factor for NSCLC patients under pembrolizumab-based immunotherapy. Future studies must determine whether withdrawal of benzodiazepines or neutralization of DBI improves the clinical response to immunotherapy.