Characterization of materials developed for medical usage frequently includes studies in which the materials are inoculated with bacteria in order to assess bacterial colonization and biofilm formation. Observed differences in bacterial growth are typically considered to be due to the material or the incubation conditions. To our knowledge, the method used to prepare the materials has generally not been considered with regard to its influence on bacterial colonization. The objective of this study was to determine the effects that various preparation methods exert on bacterial colonization of polymer disks. Polymer disks of the same dimethacrylate composition were photopolymerized: (1) between untreated glass slides, (2) between polyester release film, (3) between glass slides treated with an alkyl silane, (4) between glass slides treated with a perfluorinated silane, or (5) with one free surface in an argon-purged chamber. Surface chemistry was quantified using X-ray photoelectron spectroscopy, hydrophobicity was assessed by water contact angle, and topography was characterized using atomic force microscopy. The disks were inoculated with Streptococcus mutans for 4 h, fixed, and visualized using confocal laser scanning microscopy. Differences among all groups were found with regard to surface chemistry, hydrophobicity, topography, and bacteria morphology, density, and coverage, indicating that the method of sample preparation strongly affects both the surface properties and the initial bacterial colonization. Polymerization on untreated slides was selected as the preferred method of preparation due to minimal material transfer to the polymer and consistent, reproducible bacterial colonization.