Background: Urinary tract stone (UTS) is a common disease significantly impacting human health. Obesity influences stone formation and increases UTS incidence, yet the differences in the urinary microbiota and pathways between overweight and healthy-weight UTS patients remain unclear. Methods: In this study, 16 patients were analyzed: 8 overweight and 8 healthy-weight UTS patients. Bladder urine samples were collected during surgery, and DNA was extracted for microbial analysis using 2bRAD markers. Microbial diversity and KEGG pathway differences were studied. Results: The results showed that overweight UTS patients had a significantly higher urinary microbial diversity than healthy-weight patients. The analysis identified differences in microbiota at various taxonomic levels. LEfSe analysis revealed Sphingomonas_paucimobilis as abundant in overweight patients, while Bifidobacterium_piotii dominated in healthy-weight patients. Key species, including Ralstonia_sp000620465, Sphingomonas_paucimobilis, and Campylobacter_D_coli, were identified. KEGG analysis highlighted enriched pathways in overweight UTS patients, including the porphyrin and chlorophyll metabolism, fatty acid metabolism, amino acid degradation, and renin–angiotensin and mineral absorption pathways. Conclusions: This study is the first to use 2bRAD-M microbiome analysis to compare the urinary microbiota between overweight and healthy-weight UTS patients. It identified significant microbiota and pathway differences, suggesting a link between microbiota imbalance, obesity, and stone formation. These findings provide potential targets for further research on obesity-related stone susceptibility mechanisms.

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