Context: Safe food is a critical issue in the prevention of foodborne diseases. Food handlers play an essential role in the prevention of foodborne diseases and food poisoning at all stages of food preparation, storage, and handling. Aim: The study aimed to evaluate the effect of hygiene guidelines on knowledge, attitudes, and practices of food handlers at University cafeterias. Methods: A quasi-experimental design used to carry out the study. Setting: The study conducted at the Faculties Cafeteria at Benha University. A convenient sample of (60) food handlers worked at Cafeterias of Benha University. Three tools used for collecting data: A Structured interview questionnaire included two parts. First is concerned with the assessment of the food handlers' demographic characteristics. Second part is concerned to assess knowledge, and the third part included assessment of food handlers' practices of the food handlers about food hygiene. The second tool is food handlers' attitude assessment scale. The third tool is an environmental observational checklist assessed the cafeteria's environmental condition. Results: 73.3% of the studied food handlers were male and single, 50.0% of them aged 20 - <30 years with mean± SD (32.63±4.71). Additionally, 76.7% of food handlers had a secondary education level, and they had health certificates. Only 18.3% of the food handlers had a good knowledge regarding food hygiene before the guideline sessions compared to 85.0% after the guideline sessions. On the other hand, 65.0% of food handlers had a negative attitude toward food hygiene before the guideline sessions decreased to 15.0% after the guideline sessions. Moreover, 68.3% of food handlers had unsatisfactory practices regarding food hygiene before the guideline sessions compared to 76.7% after the guideline sessions. Conclusion: A considerable improvement noticed among the studied food handlers after the guideline sessions related to the knowledge, attitude, and practices of food hygiene. Recommendations: Further research should be geared towards regularly implementing health education programs for food handlers about food safety and hygiene at all faculties of Egypt universities.