Biofilms, structured communities of microorganisms encased in an extracellular matrix, are a major cause of persistent infections, particularly when formed on medical devices. This study investigated the kinetics of biofilm formation by Escherichia coli and Pseudomonas aeruginosa, two clinically significant pathogens, on two medical-grade polymers: polyether ether ketone (PEEK) and polyamide 12 (PA12). Using a modified crystal violet staining method and spectrophotometric quantification, we evaluated biofilm development over time on polymer granules and catheter segments composed of these materials. Results revealed that PEEK surfaces supported significantly more biofilm formation than PA12, with peak accumulation observed at 24 h for both pathogens. Conversely, PA12 demonstrated reduced bacterial adhesion and lower biofilm biomass, suggesting surface characteristics less conducive to microbial colonization. Additionally, the study validated a reproducible protocol for assessing biofilm formation, providing a foundation for evaluating anti-biofilm strategies. While the assays were performed under static in vitro conditions, the findings highlight the importance of material selection and early prevention strategies in the design of infection-resistant medical devices. This work contributes to the understanding of how surface properties affect microbial adhesion and underscores the critical need for innovative surface modifications or coatings to mitigate biofilm-related healthcare risks.