Complex multispecies biofilms consistently colonize the interior of indwelling urinary catheters, causing persistent asymptomatic bacteriuria and frequent symptomatic episodes in long-term catheterized individuals (1). Simple single-species models often fail to capture the complexities of mixed-species interactions, leading to misleading conclusions about microbial behaviour and treatment efficacy. Additionally, using lab-based organisms can obscure the genomic diversity found in real-world infections. The primary objective of this study was to establish a stable and reproducible in vitro biofilm model derived from the multi-species clinical flora associated with catheter-related infections, reflecting the dynamics of in vivo infections. Biofilm samples from clinical catheters of spinal cord injury (SCI) participants were used to establish polymicrobial macro-fluidic models within catheters. Metagenomic techniques using short-read Illumina and long-read Oxford Nanopore sequencing was used to assess the community composition, strain-level phylogeny diversity and SNP analysis of isolates. Antibiotic resistance tests using our models highlighted the drastic differences between planktonic bacteria, single-species, and multispecies biofilms. In silico analysis of antibiotic resistance revealed an alarmingly high number and varied resistance genes present in these communities. These models were developed and characterized in this study as they can be crucial for devising effective strategies to prevent and treat catheter-associated infections.