Probiotics may improve mood, but their role as adjunctive therapy for major depressive disorder (MDD) is not well understood. This study examines the effects of probiotics on depression severity, brain-derived neurotrophic factor (BDNF), adrenocorticotropic hormone (ACTH), and cortisol levels in MDD patients. Fifty medication-free MDD patients were randomized to receive probiotics with fluoxetine ( = 25) or placebo with fluoxetine ( = 25) for 8 weeks. Depression severity was assessed using the Hamilton Depression Rating Scale (HDRS-24), and fasting blood samples were collected at baseline and study conclusion. Forty-four patients completed the trial. The probiotic group showed a significant reduction in depression severity compared with the placebo group ( = 0.001). No significant differences were observed in serum cortisol ( = 0.46) and ACTH levels ( = 0.44). Plasma BDNF levels increased slightly in the probiotic group but were not statistically significant. Probiotic supplementation with fluoxetine significantly reduces depression severity in MDD patients.