Male lower urinary tract symptoms (LUTS) comprise a common syndrome of aging that negatively impacts quality of life. The etiology of LUTS is multifactorial, involving benign prostatic hyperplasia, smooth muscle and neurologic dysfunction, inflammation, sexually transmitted infections, fibrosis, and potentially dysbiosis, but this aspect remains poorly explored. We investigated whether the presence of infectious agents in urine might be associated with LUTS by combining next-generation DNA sequencing for virus discovery, microbiome analysis for characterization of bacterial communities, and mass spectrometry-based metabolomics. In urine from 29 LUTS cases and 9 controls from Wisconsin, we found a statistically significant association between a diagnosis of LUTS and the presence of JC virus (JCV), a common neurotropic human polyomavirus (Polyomaviridae, Betapolyomavirus) linked to severe neurologic disease in rare cases. This association (based on metagenomics) was not borne out when specific polymerase chain reaction (PCR) testing was applied to this set of samples, likely due to the greater sensitivity of PCR. Interestingly, urine metabolomics analysis identified dysregulation of metabolites associated with key LUTS processes. Microbiome analysis found no evidence of microbial community dysbiosis in LUTS cases, but JCV-positive samples contained more Anaerococcus species, which are involved in polymicrobial infections of the urinary tract. Neither age nor body mass index were significantly associated with the presence of urinary JCV-in the initial group or in an additional, regionally distinct group. These data provide preliminary support the hypothesis that viruses such as JCV may play a role in the development or progression of LUTS, together with other infectious agents and host metabolic responses.