It is well known that the common goal of all central sterile supply departments (CSSDs) is to prevent healthcare-associated infections. Such infections entail high costs to society, not only economic but also social. Therefore, delivering safe medical devices and guaranteeing a positive contribution to the control of healthcare-associated infections form the main responsibilities of a CSSD. The monitoring of the effectiveness of medical device cleaning processes is highly recommended. However, ensuring a flawless environment for the preparation, assembly, and packaging of medical devices and clean handling of sterilized items is crucial to achieving the goal of safe medical devices. This study analyzed not only the cleanliness of surgical instruments but also two critical aspects of the surrounding environment: the cleanliness of work surfaces and the cleanliness of workers’ hands. To evaluate the cleanliness of surgical instruments, two methods were used: the adenosine triphosphate (ATP) detection method and a residual protein test. It was not the intention of this work to make an exhaustive comparison of these methods. The ATP bioluminescence method was also used for monitoring the cleanliness of work surfaces and workers’ hands. The aims of this study were to establish the most suitable method of evaluating the cleanliness of reusable medical devices in the CSSD and to assess the quality of the environment. Assessing the surgical instruments, work surfaces, and staff hands for cleanliness allowed the identification of possible contamination sources and to correct them by improving cleaning/disinfection protocols. Furthermore, the use of ATP monitoring tests of workers’ hands highlighted the importance of staff compliance with good practice guidelines. Thus, these results have a positive impact on the CSSD quality system and, consequently, on patient safety.