This study evaluated the knowledge, attitudes, and practices (KAP) of Central Sterile Supply Department (CSSD) staff at Ministry of Defence hospitals in the Eastern Province of Saudi Arabia regarding sterilization protocols. A cross-sectional study was conducted using a self-administered online questionnaire distributed to 54 CSSD staff members. The questionnaire assessed demographic characteristics, knowledge, attitudes, and practices related to sterilization. KAP scores were categorized as poor, moderate, or good. Results: The findings indicated that 57.4% of participants had adequate knowledge, 55.6% adhered to good sterilization practices, and 24.1% displayed a positive attitude toward sterilization. A positive correlation was found between knowledge and attitude (rs=0.348, p=0.010) and practice scores (rs=0.302, p=0.027). However, some knowledge gaps were identified in areas such as spill kit use and the distinction between scopes. While the majority of CSSD staff demonstrated adequate knowledge and practice, there is a need for targeted training to address knowledge gaps and improve attitudes, particularly regarding specific sterilization practices. Hospitals should prioritize continuous education and targeted training programs for CSSD staff to enhance sterilization protocols, thereby improving infection prevention and control measures. Further research is needed across a broader range of hospitals to assess national trends.