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Background Gut microbiome dysbiosis is increasingly recognized as a key contributor to the pathogenesis of Irritable Bowel Syndrome (IBS), influencing intestinal permeability, immune activation, and gut-brain signaling. Probiotics have emerged as a therapeutic option, although clinical responses remain heterogeneous. In particular, whether multi-strain formulations confer superior benefit compared to single-strain probiotics remains uncertain. Evidence from Southeast Asian populations is rare, despite distinct dietary patterns and microbiome profiles that may influence treatment response. This study aimed to evaluate the comparative real-world effectiveness of multi-strain versus single-strain probiotics in patients with IBS.Methods The researcher conducted a prospective real-world cohort study at Bali International Hospital, Indonesia, between September 2025 and February 2026. Adult patients fulfilling Rome IV criteria for IBS were consecutively enrolled. Patients received either single-strain or multi-strain probiotics for 8 weeks, based on the physician’s discretion and routine clinical practice. The primary outcome was the change in IBS Severity Scoring System (IBS-SSS). Secondary outcomes included abdominal pain scores and IBS Quality of Life (IBS-QoL). Clinical response was defined as a reduction of ≥50 points in IBS-SSS. Exploratory subgroup analyses were performed according to IBS subtype (IBS-D, IBS-C, IBS-M). Descriptive and comparative analyses were conducted to evaluate treatment outcomes.Results A total of 64 patients were included (mean age 39.6±12.1 years; 59% female). Patients receiving multi-strain probiotics demonstrated a greater reduction in IBS-SSS compared to those receiving single-strain therapy (−104.2 vs −65.8), corresponding to a clinically meaningful difference in symptom burden. Clinical response (≥50-point reduction) was observed in 68.8% of patients in the multi-strain group compared to 46.9% in the single-strain group. Improvements in abdominal pain and IBS-QoL followed a consistent pattern favoring multi-strain therapy. The magnitude of benefit appeared more pronounced among patients with diarrhea-predominant IBS. No serious adverse events were reported.Conclusions In this prospective real-world study, multi-strain probiotics were associated with greater and clinically meaningful improvement in IBS symptoms compared to single-strain formulations. Despite the modest sample size, the consistent direction and magnitude of effect across multiple outcomes support a potential therapeutic advantage of multi-strain probiotics. These findings provide region-specific evidence from Southeast Asia and warrant confirmation in larger, adequately powered studies.