A surgical site infection (SSI) is one of the most common surgical complications. This study analyzed different sources of microorganisms in the air, on reusable surgical instruments, and the outer surface of sterile packaging systems during the use and reprocessing of sterile goods (from the operating room (OR) to the Central Sterile Supply Department (CSSD)). The microbial load in the air was analyzed via active air sampling and settle plates. Furthermore, the airborne particle load was measured by a particle counter. Contact agar plates were used to determine the microbial load on surgical instruments and sterile packaging systems. The highest average microbial and particle load was measured in the air of the OR (active air sampling: max. 56 CFU/m3; settle plates: max. 9 CFU; ≥0.3 μm particles in size: 1,958,403 no./m3). However, no microbial load (0 CFU) was detected on surgical instruments sampled in the OR. The outer surface of stored sterile packaging systems showed a maximal microbial load of 64 CFU. The most common identified pathogen was coagulase-negative staphylococci. Compared to properly reprocessed reusable surgical instruments and sterile packaging systems, the air still seems to be the primary potential source of microbial contamination, especially within the OR.