The reduction of biofouling and the reduction of cross-contamination in the food industry are important aspects of safety management systems. Polymeric surfaces are used extensively throughout the food production industry and therefore ensuring that effective cleaning regimes are conducted is vital. Throughout this study, the influence of the surface characteristics of three different polymeric surfaces, polytetrafluoroethylene (PTFE), poly(methyl methacrylate) (PMMA) and polyethylene terephthalate (PET), on the removal of Escherichia coli using a wipe clean method utilising 3% sodium hypochlorite was determined. The PTFE surfaces were the roughest and demonstrated the least wettable surface (118.8°), followed by the PMMA (75.2°) and PET surfaces (53.9°). Following cleaning with a 3% sodium hypochlorite solution, bacteria were completely removed from the PTFE surfaces, whilst the PMMA and PET surfaces still had high numbers of bacteria recovered (1.2 × 107 CFU/mL and 6.3 × 107 CFU/mL, respectively). When bacterial suspensions were applied to the surfaces in the presence of a blood conditioning film, cleaning with sodium hypochlorite demonstrated that no bacteria were recovered from the PMMA surface. However, on both the PTFE and PET surfaces, bacteria were recovered at lower concentrations (2.0 × 102 CFU/mL and 1.3 × 103 CFU/mL, respectively). ATP bioluminescence results demonstrated significantly different ATP concentrations on the surfaces when soiled (PTFE: 132 relative light units (RLU), PMMA: 80 RLU and PET: 99 RLU). Following cleaning, both in the presence and absence of a blood conditioning film, all the surfaces were considered clean, producing ATP concentrations in the range of 0–2 RLU. The results generated in this study demonstrated that the presence of a blood conditioning film significantly altered the removal of bacteria from the polymeric surfaces following a standard cleaning regime. Conditioning films which represent the environment where the surface is intended to be used should be a vital part of the test regime to ensure an effective disinfection process.