Summary

Intravesical mycobacterial immunotherapy is a standard treatment for non-muscle-invasive bladder cancer (NMIBC), yet its impact on the urinary and intestinal microbiota and its potential implications for therapeutic efficacy remain poorly understood. This study systematically characterizes bladder and gut microbiota responses to intravesical Mycobacterium bovis BCG and the non-pathogenic immunomodulator Mycobacterium brumae in healthy and tumor-bearing mice. We show that intravesical mycobacterial therapy induces site- and treatment-specific alterations in microbial diversity and community structure, affecting bacterial taxa in both bladder and gut. BCG and M. brumae elicited distinct microbiota signatures, while tumor status further shaped treatment-associated microbial profiles.These findings identify intravesical mycobacterial therapy as a modulator of microbial ecosystems beyond the site of administration and provide new insights into the host–tumor–microbiota–immunotherapy axis. Understanding these interactions may enable the identification of microbiome-associated biomarkers and support the development of more effective and personalized bladder cancer immunotherapies.