Background: Several randomized controlled trials (RCTs) have shown conflicting results on cortisol levels following probiotic administration in healthy and diseased populations. Previous analyses were inconclusive due to limited studies, and evidence is lacking on how these effects vary by health status; region; therapy duration; medications, and use of single or multiple strains. Methods: In this systematic review and meta-analysis (PROSPERO [CRD42024538539]), we searched PubMed, Cochrane Library, Embase, Scopus, Web of Science, CINAHL, ProQuest, and Web of Science Preprints until 13 August 2024, for RCTs on probiotic administration, either alone or combined, across all age groups and without specific medical condition requirements. We applied random-effects meta-analysis, assessed bias using the Cochrane RoB 2 tool, and evaluated evidence certainty with GRADE. Findings: We screened 1739 records and retrieved 46 RCTs (3516 participants). Probiotics supplementation decreased cortisol levels compared to the control arm [46 RCTs; SMD: −0.45; 95% CI: −0.83; −0.07; I²: 92.5%, low certainty]. Among various subgroups; probiotics supplementation decreased the cortisol levels in the subgroups without concomitant medications [37 RCTs; SMD: −0.30; 95% CI [−0.58; −0.03], I²: 88.7%] with a single probiotic strain [30 RCTs; SMD: −0.33; 95% CI: −0.63; −0.028; I²: 88.8%], in a healthy population [35 RCTs; SMD:−0.3; 95% CI: −0.58; −0.03; I²: 88.7] and in the Asia region [21 RCTs; SMD: −0.83; 95% CI: −1.58; −0.07; I²: 95%]. Interpretation: A low level of evidence suggests probiotics might reduce cortisol levels, but more targeted studies are needed to identify variables affecting the response in specific subgroups.