When Proteus mirabilis, Enterococcus faecalis, and Escherichia coli are co-cultured in human urine, they form biofilms with increased biomass. This increase in biomass is due to a rise in the total protein content of the biofilm rather than other components, such as carbohydrates or nucleic acids. The study focused on identifying proteins that are enriched in polymicrobial biofilms (co-cultures) compared to monomicrobial biofilms. Proteins that increase in relative abundance during co-culture may serve as targets for preventing the formation of recalcitrant polymicrobial biofilms.