Purpose <p>Antidepressant use, which targets intracerebral serotonin metabolism, is common among glioblastoma patients. However, its interaction with tumor metabolism and survival remains unclear. We investigated the relationships between antidepressant use, intratumoral serotonin pathway metabolites, quality-of-life, and survival.</p> Methods <p>We analysed two complementary cohorts: a population-based cohort (<i>n</i> = 801) and a hospital-based biobank cohort (<i>n</i> = 153). In the population-based cohort, information about antidepressant use and survival were obtained from national registers. In the hospital-based cohort, intratumoral serotonin pathway metabolites were measured, and data on preoperative antidepressant use and quality-of-life were recorded. Linear and Cox regression models were used to investigate the association between antidepressant use, metabolites, quality-of-life and survival.</p> Results <p>In the population-based cohort, antidepressant use was associated with poorer survival in adjusted analyses. However, use of fluoxetine or sertraline was associated with better survival compared with other selective serotonin reuptake inhibitors (HR = 0.62, 95% CI = 0.44–0.88). In the hospital-based cohort, preoperative antidepressant use was associated with lower intratumoral serotonin levels and its downstream metabolite, 5-HIAA. Higher serotonin levels were associated with better preoperative quality-of-life, especially general health. Serotonin pathway metabolites were not clearly associated with survival.</p> Conclusions <p>These findings suggest that higher tumor tissue serotonin abundance was associated with better patient-reported well-being but not with survival in gliobastoma. Survival differed across SSRI exposure groups, although causal interpretation is limited by the observational design. These findings do not provide strong evidence against the continued clinical use of sertraline or fluoxetine in glioblastoma patients when indicated.</p>