Medical devices are a vital part of the global health care system that can have a far-reaching impact on patient treatment. Therefore, they must be sterile to ensure patient safety. The prevalent microorganism's type on a medical device, also known as "bioburden", is a useful indicator of a potential contamination source. Indeed, bioburden is a potential risk to the patient not only because the sterilization process might not be completely effective, but also post-processing because of the possible presence of residual materials. Although bioburden may be confidently killed by destructive sterilization processes, its proliferation before sterilization should be avoided. For the bioburden determination, the culture media and incubation conditions must be carefully selected. The culture medium is of fundamental importance for most microbiological tests: to obtain pure cultures, to grow and count microbial cells, and to cultivate and select microorganisms. A culture medium is essentially composed of basic elements (water, nutrients) to which must be added different growth factors that will be specific to each bacterium and necessary for their growth. Without high-quality media, the possibility of achieving accurate, reproducible, and repeatable microbiological test results is reduced. In ISO 11737-1:2018 "Sterilization of Health Care Products-Microbiological Methods-Part 1: Determination of a Population of Microorganisms on Products", methods for the determination and microbial characterization of bioburden are proposed. However, few guidelines are given as to culture media other than examples and incubation times. Several studies show that other culture media can also be effective, such as Plate Count Agar (PCA). The purpose of this review was to focus on parameters that can have an impact on the bioburden evaluation, specifically the culture medium type for the microorganisms' detection on medical devices. Experimentations conducted in our laboratories showed that PCA appeared to be a medium of primary importance for the detection of bioburden on medical devices; this medium also respects the 3Rs rule.