Biomaterials consist of both natural and synthetic components, such as polymers, tissues, living cells, metals, and ceramics. Their purpose is focused on repairing or replacing malfunctioning living tissues and organs. Therefore, it is imperative to ensure the safety and sterility of biomaterials before any contact with living tissue. Ultraviolet (UV)-C irradiation for biomaterial disinfection has been considered due to the high recurrence rate of bacterial infections and to prevent resistance. Physical composition and surface properties and UV-C sensitivity of microorganisms can alter its efficacy. The main objective of this study was to evaluate the efficacy of UV-C in terms of microbial lethality and additional underlying factors contributing to its performance, namely the surface properties. For this purpose, twelve different strains were first screened, from which four microorganism species known to have the ability to cause nosocomial infections were further tested, namely Escherichia coli, Pseudomonas aeruginosa, Candida albicans, and Candida glabrata. These microorganisms were inoculated onto slides and disks of various bio contact surfaces, including glass (GLS), titanium (Ti), and poly ether etherketone (PEEK), and exposed to UV-C. The results demonstrate that bacterial pathogens on biomaterial surfaces respond differently to UV-C light exposure, and the bactericidal effect decreased in this order: glass, PEEK, and Ti (0.5 to 2.0 log reduction differences). P. aeruginosa ATCC 27853 on glass surfaces was reduced to an undetectable level after being exposed to 6.31 J.cm−2 of UV-C, displaying the highest reduction rate observed among all the tested microorganisms, 2.90 J−1.cm−3, compared to Ti and PEEK. Similarly, a higher reduction in C. glabrata ATCC 2001 was observed on glass; the modeled inhibition displayed a rate of 1.30 J−1.cm−3, the highest observed rate among yeast, compared to Ti and PEEK, displaying rates of 0.10 J−1.cm−3 and 0.04 J−1.cm−3, respectively. The inactivation rates were higher for less hydrophobic materials with smoother surfaces as compared to biomaterials with rougher surfaces.