Background: The misuse of antibiotics could result in a public health problem including the high prevalence of antibiotic resistance. Objectives: Assessing the knowledge, attitude and practice (KAP) regarding the antibiotics misuse in Kingdom of Saudi Arabia (KSA). Methods: A cross-sectional questionnaire based study was conducted at AL-Mahd General Hospital, Saudi Arabia, Ministry of Health, from February to June 2017 among 721 adult Saudi subjects. Results: The most common source of knowledge about antibiotics among participants was a physician (50.8%) followed by internet in 25.9%. About 50.3% of subjects used antibiotics more than 5 times per year and 65.6% of participants have recently used antibiotics. Insufficient knowledge was found among 59.6% of respondents and most of the subjects declared negative attitude and poor practice pattern. The good knowledge was associated with being young, female and higher educational degree. Conclusion: The level of knowledge was insufficient and resulted in inappropriate attitude and practice pattern in KSA. The lower educational degree, young age and female gender were significant contributors to sufficient knowledge about antibiotic use.