Actinotignum massiliense, a Gram-positive, facultatively anaerobic coccoid rod, is a rare human pathogen able to infect the urinary tract and belongs to the order of Actinomycetales. We identified A. massiliense as a resident of microbial biofilms growing on indwelling urethral catheter surfaces that were isolated from two patients with neurogenic bladders. These catheter biofilms (CBs) also harbored common uropathogens such as Proteus mirabilis and Aerococcus urinae, supporting the notion that A. massiliense depends on other co-colonizing microbes for survival. We isolated the bacterium from an anaerobically grown culture of a clinical sample, identified the species by 16S rRNA gene sequencing and verified this result via shotgun proteomics. Bacterial proteomes were profiled from the in vitro grown strain and four clinical ‘in vivo’ samples. The quantified proteomes allowed us to infer metabolic pathways and virulence/survival factors of importance in the CB milieu. Two putative subtilisin-like proteases, two Rib/Esp surface antigen repeat-containing proteins, a papain-like cysteine protease and a metal/heme/oligopeptide uptake system were highly expressed in vivo, but less so in vitro. We predict these proteins to be critical for adhesion and growth in CBs attacked by the host’s innate immune system or to improve bacterial fitness. Mixed acid fermentation following uptake and metabolism of xylose and glucuronate, sugars highly represented in proteoglycans and glycoglycerolipids of the urothelial mucosa and, in the case of glucuronate, shed into urine via renal xenobiotic conjugates, is inferred to be a major pathway for A. massiliense to generate energy under microaerobic conditions in CBs. The bacteria also appear to have active pathways for storage and utilization of glycogen as a carbon resource. Finally, we identified a putative polyketide synthase which may generate a secondary metabolite that interacts with either the host or co-colonizing organisms to enable A. massiliense survival in CBs.