Background: Barber’s shop is a source of infections agents both blood borne and other infestation and the sure way to prevent this is through instrument sterilization after each customer. Aim: The study was to determine the level of knowledge and the practice of instrument sterilization amongst barbers in Presco campus, Ebonyi State University Abakaliki. Materials and Method: Cross-sectional design was used in the study. The sample of 40 barbers was drawn using simple sampling technique. Data was collected using questionnaire and analyzed using frequency and percentage, and ANOVA. Result: Majority (87.5%) were trained by other barbers as apprentice, very many (75%) were licensed to practice barbing, only 10 (25%) had accurate knowledge and aware of the need to sterilize instrument after use on each customer, personal characteristics have no significant influence on participant’s knowledge of instrument sterilization at p=0.05. Conclusion: Barbers need proper education on the appropriate instrument sterilization to limit the spread of deadly infectious diseases.