Background: hand hygiene is now regarded as one of the most important element of infection control activities. It is an effective strategy to prevent health care-associated infections (HCAIs). It is a required practice for all health care providers. It is done by using manually antibiotics jelly or washing hands by soap and tap water. Hand hygiene before and after examination of the patient is the first line of protection against infection as hand is considered the primary carrier of infections. Aim of the Work: was to assess the knowledge, attitudes and practice of doctors and nurses in King Abdulaziz Specialist Hospital. Patients and Methods: this descriptive cross-sectional study was conducted on the 110 of doctors and nurses in King Abdulaziz Specialist Hospital, who were interviewed between 3rd March 2017 and 9thApril 2017. Data were collected and analyzed using SPSS version 16. Results: out of 110 respondents, 57.3% were female and the nurses constituted the highest cadre (61.8%). Hand hygiene knowledge was highest in the Emergency Room (29.1%) and  lowest among diabetes center staff (3.6%). Conclusion: hand hygiene is crucial for doctors and nurses and hence the need to improve their attitude, practice and knowledge. The presence of significant differences between doctors and nurses in our study indicated the need for qualified doctors who have enough information and knowledge about the importance of hand hygiene to assure control of infections as well as all hygienic affairs during working with patients enrolled in hospitals.