Background The Central Sterile Supply Department (CSSD) plays a crucial role in preventing healthcare-associated infections through proper instrument reprocessing. However, critical control points in the reprocessing workflow and their direct association with surgical site infection (SSI) remain inadequately characterized. Objective To identify key control points in CSSD instrument reprocessing and analyze their association with surgical site infection (SSI) rates, defined according to Centers for Disease Control and Prevention (CDC) criteria through a comprehensive retrospective analysis. Methods A retrospective cohort study was conducted from January 2020 to December 2023 at a tertiary care hospital. We analyzed 15,847 surgical procedures and corresponding CSSD reprocessing records. Key control points were identified through process mapping, failure mode analysis, and statistical correlation with infection outcomes. Multivariate logistic regression was used to determine independent risk factors. Results Seven critical control points were identified: pre-cleaning verification (OR: 2.34, 95% CI: 1.78–3.09), cleaning efficacy validation (OR: 3.12, 95% CI: 2.45–3.98), sterilization parameter monitoring (OR: 4.67, 95% CI: 3.89–5.61), sterile integrity maintenance (OR: 2.89, 95% CI: 2.21–3.78), staff competency verification (OR: 2.56, 95% CI: 1.99–3.29), equipment maintenance compliance (OR: 3.45, 95% CI: 2.67–4.46), and quality documentation completeness (OR: 1.98, 95% CI: 1.54–2.54). Overall SSI rate was 2.4% (380/15,847 procedures). Inadequate control at identified points increased infection risk by 180–367%. Conclusion Seven key control points are significantly associated with SSI rates. Implementation of enhanced monitoring and control measures at these points may be associated with SSI and improve patient safety outcomes.