There is increasing evidence that the hospital surface environment contributes to the spread of pathogens. However, evidence on how best to sample these surfaces is inconsistent and there is no guidance or legislation in place on how to do this. The aim of this review was to assess current literature on surface sampling methodologies, including the devices used, processing methods, and the environmental and biological factors that might influence results. Studies published prior to March 2019 were selected using relevant keywords from ScienceDirect, Web of Science, and PubMed. Abstracts were reviewed and all data-based studies in peer-reviewed journals in the English language were included. Microbiological air and water sampling in the hospital environment were not included. Although the numbers of cells or virions recovered from hospital surface environments were generally low, the majority of surfaces sampled were microbiologically contaminated. Of the organisms detected, multidrug-resistant organisms and clinically significant pathogens were frequently isolated and could, therefore, present a risk to vulnerable patients. Great variation was found between methods and the available data were incomplete and incomparable. Available literature on sampling methods demonstrated deficits with potential improvements for future research. Many of the studies included in the review were laboratory-based and not undertaken in the real hospital environment where sampling recoveries could be affected by the many variables present in a clinical environment. It was therefore difficult to draw overall conclusions; however, some recommendations for the design of routine protocols for surface sampling of healthcare environments can be made.