Several urinary disorders, including overactive bladder, urinary incontinence, and interstitial cystitis, are often characterized by negative urine cultures. The application of metagenomics (i.e., 16S rRNA microbial profiling or whole-genome shotgun sequencing) to urine samples has enabled the identification of previously undetected bacteria, contributing to the discovery and characterization of the urinary microbiome. The most frequent species isolated are Lactobacillus (15%), Corynebacterium (14.2%), Streptococcus (11.9%), Actinomyces (6.9%), and Staphylococcus (6.9%). Although several studies are emerging in this context, the role of urinary microbiota in the pathogenesis of infections and in tumor carcinogenesis remains unclear. Furthermore, data on the activity of gut microbiota in modulating sensitivity to immune checkpoint inhibitors in advanced cancer patients suggest that the influence of urinary microbiota on tumor response to anticancer therapy should also be investigated. Moreover, its possible relationship with tumor mutational burden, which is in turn correlated with response to immunotherapy, should be the focus of future studies. Of note, the effect of antibiotics on this complex scenario seems to deserve careful consideration.