Purpose: The effect of knowledge and practice of health workers (HWs) on surgical site infection (SSI) has never been documented in our area and such knowledge gaps has made advocating for allocation of resources to tackle the vice of SSI and its menacing outcome challenging hence this study. Method: A cross-sectional descriptive study on randomly selected 60 health care workers from Lira regional referral hospital (LRRH) and Lira university teaching hospital (LUTH) was conducted for 1 month. Data was entered and analyzed descriptively and inferentially using SPSS version 23. Results: Amongst the 60 respondents enrolled, the study found that practicing infection prevention method is the single most significant predictor of SSI prevention compared to just possession of knowledge by the HWs (P value 0.003). Further to that, approximately 7.3% of health workers don’t practice measures required to prevent surgical site infections, 12.51% of them lack the requisite knowledge on prevention of SSI of which anesthetic officer and nurses were the most likely category of health employees to have poor practice 33.3% and 27.3% respectively. Conclusion: A significant number of HWs have limited knowledge and other do not practice all measure required in prevention of SSI, hence, the need for more practical sessions in training institutions