Research indicates that polymicrobial urinary tract infections (UTIs) are relatively common, accounting for up to ~40% of UTIs. These infections can be more detrimental to the host than those involving only a single species. Escherichia coli is the most important agent in UTIs, and Enterococcus faecalis is the second or third most common uropathogen, depending on the patient population. The co-existence of E. coli and E. faecalis has been observed in a significant proportion of UTIs. Polymicrobial infections involving E. coli and E. faecalis are associated with a high frequency of recurrence, emphasising the clinical significance of these co-infections. Candida albicans is the most common fungal uropathogen, being more frequent in certain patient populations, including those with frequent antibiotic use, such as in recurrent UTIs. The formation of biofilms by E. coli, E. faecalis, and C. albicans is a significant factor in the pathogenesis and management of UTIs. Biofilms can contribute to antimicrobial resistance, persistent infections, and polymicrobial interactions, posing challenges for treatment and eradication. This study aimed to develop a clinically relevant in vitro triple-species biofilm protocol for UTI research, as the extrapolation of data from monomicrobial to polymicrobial biofilms is limited. Reproducible triple-species biofilms were established in 96-well polystyrene plates in CAMHB medium at 37°C, 5% CO2 for 24 hours. Biofilms were disrupted for 4-6 hours with either ethylenediaminetetraacetic acid (EDTA) (25mM), ciprofloxacin (0.5µg/mL, 5µg/mL and 25µg/mL), fluconazole (2µg/mL, 20µg/mL and 100µg/mL) or N-acetyl-cysteine (NAC) (1.25mg/mL, 2.5mg/mL, 5mg/mL and 10mg/mL). Biofilm disruption was determined by measuring biofilm biomass via crystal violet assay and cell viability via colony-forming unit (CFU) per millilitre (CFU/ml). The treatment effects on different microorganisms within the polymicrobial biofilms varied greatly, highlighting the complexity of interactions within these biofilm communities. Only one treatment, ciprofloxacin, significantly reduced the biofilm biomass and E. coli viability at all concentrations. The protocol could be useful for the study of interkingdom relationships and assist in finding new strategies against polymicrobial recurrent UTIs.