Background: Fungal contamination of surgical instruments poses a significant risk to patient safety, potentially leading to surgical site infections and other severe complications. By examining contamination levels both immediately after opening sterile sets and after two hours of environmental exposure, it becomes possible to assess contamination reduction strategies. Objectives: The aim of this study was to identify which instruments and operating rooms were most affected by fungal contamination. Understanding the extent of this contamination is critical to developing more effective sterilization protocols, improving infection control practices, and ultimately enhancing patient safety in surgical settings. Methods: This study was conducted on surgical instruments at Ali ebn Abi Talib Zahedan Hospital in 2023, across seven operating rooms (emergency, general surgery, gynecology, and urology). Samples were randomly collected under sterile conditions to prevent external contamination. The collected samples were cultured in Sabouraud Dextrose Agar (SDA), and data were analyzed using SPSS version 26. Results: The findings revealed that different surgical instruments, such as Pinzette (without teeth), Allis forceps, Babcock forceps, curved clamps, Gully pots, receivers, retractors, ring forceps, and towel clamps, were contaminated with fungal pathogens within two hours of opening the sterile sets. The highest contamination rates were observed in general surgery (39%), followed by emergency (29%), urology (21%), and gynecology (11%) rooms. The most common fungal contaminants were Aspergillus spp., yeast, and Penicillium spp. Conclusions: This study highlights the challenge of maintaining sterility in surgical environments post-sterilization, as fungal contamination reappeared on surgical instruments within two hours despite the initial sterilization. These findings emphasize the need for enhanced infection control measures, advanced environmental controls, and regular monitoring to reduce the risks associated with fungal contamination in surgical settings.