Background: Hand hygiene (HH) is a general concept of practicing hand washing, either with alcohol-based hand, or with soap and water, and considered the most effective, simple and cost-effective way to prevent transmitting the infections. Objectives: To assess the knowledge, attitudes and practices of proper hand hygiene technique among family medicine residents working in Makkah, Saudi Arabia. Methods: Analytical cross-sectional study was carried out among Family medicine residents working at joint program of family medicine in Makkah city, Saudi Arabia, during 2020. A valid self-administered questionnaire in English consisted of 4 parts (demographic data, questions to assess knowledge, attitude and practice of hand hygiene) was used. Chi-square and Student t-test were used in analysis utilizing Statistical Package for the Social sciences (SPSS) version 26.0. Results: A total of 163 family medicine residents were included in the study. Overall, 87.1% and 92.6% of the family medicine residents had good level of knowledge and positive attitude, respectively towards hand hygiene. Most of them (81%) had good practice-related to hand hygiene. Older physicians were more likely to have good level of practice. Age of family medicine residents who expressed good level of practice –related to hand hygiene was significantly higher than that of those with poor level of practice (28.0±2.1 vs. 27.2±1.3), p=0.005. Conclusion: Majority of the family medicine residents had good knowledge and practice as well as positive attitude towards hand hygiene. However, knowledge and attitude towards hand hygiene were not related to its practice among them.