Abstract Context: Zoonotic diseases are infectious diseases that naturally transmitted from animals to humans. Aims: The study aimed to evaluate the effectiveness of Zoonotic Diseases prevention program for Veterinary workers in Rural Health Units at Benha City. Methods: A quazi-experimental design. The study was carried out in Veterinary Health Units at Benha City. A convenience sample used which includes (85) Veterinary workers. Two tools used for collecting data: 1) A structured interviewing questionnaire to assess: a) demographic characteristics, b) medical history of previous exposure to Zoonotic diseases and c) knowledge of Veterinary workers regarding zoonotic diseases. 2) An observational checklist sheet was used to assess: a) practices of veterinary workers regarding the prevention of zoonotic diseases, b) veterinary health unit environmental condition. Results: More than three quarters (76.5%) of veterinary workers were male, less than half (45.9%) aged ranged from 30 < 40 years with mean±SD (32.3±6.62) and about one third (31.8%) of them were secondary education. The majority (89.4%) of veterinary workers had poor total knowledge score regarding zoonotic diseases pre-program intervention compared to more than half (55.3%) of them had good total knowledge score post-program intervention. On the other hand, about two thirds (62.4%) of veterinary workers had unsatisfactory total practices score regarding zoonotic diseases pre-program intervention compared to more than three quarters (78.8%) of them had satisfactory total practices score post-program. Conclusion: Post-program score was higher than the pre-program score both in knowledge and practices of veterinary workers regarding zoonotic diseases, and there is a positive correlation between total knowledge and total practice pre and post-program (P=0.000). The study recommends an illustrated booklets and brochures in each Veterinary Health Unit focus on the use of protective methods during contact with animals and adherence to adequate sanitary standards.