Biofilms are estimated to cause approximately £45 billion in economic losses annually in the United Kingdom, with over £2 billion attributed to healthcare-associated costs. By their nature, biofilms exhibit increased resistance to desiccation and reduced susceptibility to antimicrobials, thereby posing a significant healthcare burden. Recent studies have demonstrated that biofilm-forming bacteria, such as Pseudomonas aeruginosa, can degrade plastics commonly used in healthcare environments. However, it remains unclear whether biofilm-forming bacteria preferentially colonise specific materials, or whether structural variations within the biofilm matrix facilitate attachment and persistence on different substrates. This study investigated the ‘sit and wait’ hypothesis of P. aeruginosa biofilm formation on clinically relevant materials, hypothesising that the ability to form biofilms on diverse surfaces enables environmental persistence, thereby increasing the probability of host infection. Biofilm formation by a clinical reference strain of P. aeruginosa (NCTC 13437) was assessed on polypropylene, acrylonitrile butadiene styrene, polycarbonate, stainless steel, aluminium, and copper using a crystal violet staining assay. Biofilm matrix composition and structure were further characterised using cryogenic scanning electron microscopy (Cryo-SEM), confocal laser scanning microscopy (CLSM), and light sheet fluorescence microscopy (LSFM) with fluorescent dyes targeting proteins, carbohydrates, extracellular DNA, and live/dead bacteria. Distinct differences in biofilm matrix composition and architecture were observed between materials. These findings suggest that P. aeruginosa adapts its biofilm matrix to colonise a range of clinically relevant materials, thereby facilitating environmental persistence and enhancing virulence through survival independent of immediate host infection.