The artificial urinary sphincter (AUS) is an effective treatment option for incontinence due to intrinsic sphincteric deficiency in the context of neurogenic lower urinary tract dysfunction, or stress urinary incontinence following radical prostatectomy. A subset of AUS devices develops infection and requires explant. We sought to characterize biofilm composition of the AUS device to inform prevention and treatment strategies. Indwelling AUS devices were swabbed for biofilm at surgical removal or revision. Samples and controls were subjected to next-generation sequencing and metabolomics. Biofilm formation of microbial strains isolated from AUS devices was reconstituted in a bioreactor mimicking subcutaneous tissue with a medical device present. Mean patient age was 73 (SD 10.2). All eighteen artificial urinary sphincter devices harbored microbial biofilms. Central genera in the overall microbe-metabolite interaction network were Staphylococcus (2620 metabolites), Escherichia/Shigella (2101), and Methylobacterium-Methylorubrum (674). An rpoB mutation associated with rifampin resistance was detected in 8 of 15 (53%) biofilms. Staphylococcus warneri formed greater biofilm on polyurethane than on any other material type (p < 0.01). The results of this investigation, wherein we comprehensively characterized the composition of AUS device biofilms, provide the framework for future identification and rational development of inhibitors and preventive strategies against device-associated infection.