Background: Prevention of occupational health hazards is crucial role to verify the health and safety of veterinary assistant workers in the work environment. Aim: Evaluate the effect of healthy safety program for prevention of occupational health hazards among veterinary assistant workers. Methods: A quasi- experimental design was utilized. Convenience sample of 62 veterinary assistant workers at thirty-three Veterinary Health Units in Kalyobia Governorate. Two tools were used. Tool I: An interviewing tool composed of four parts as socio-demographic characteristics, work characteristics, medical history, and knowledge of veterinary assistant workers about occupational health hazards. Tool II: Observational checklist was concerned with two parts: a- environmental work setting and b- practices of veterinary assistant workers regarding prevention of occupational health hazards. Results:58.1% of the studied veterinary assistant workers had poor knowledge regarding prevention of occupational hazards pre healthy safety program implementation, which declined to 8.1% post healthy safety program implementation, and 88.7% of the studied veterinary assistant workers had unsatisfactory practices pre healthy safety program implementation compared to 17.7% post healthy safety program implementation. Conclusion: Healthy safety program was successful in enhancing knowledge and improving health practices of veterinary assistant workers regarding prevention of occupational health hazards. Recommendations: Continuing healthy safety program for veterinary assistant workers to raise and update their knowledge and practices regarding prevention of occupational health hazards.