Background: Antibiotics are a cluster of drugs used to combat microorganisms and treat infections, hence they are the major tool to help the natural defence of the body in the management of infection. Internationally, antibiotic misuse has been reported to be due to multiple factors, including lack of compliance, and availability of antibiotics as over-the-counter medications with absence of rules and regulations that prohibit such practice. Aim: To assess knowledge, attitudes, and practices concerning self-medication with antibiotics among the public in Aseer region, Saudi Arabia. Methodology: A population based cross-sectional survey was conducted targeting the general population in Aseer region, southern Saudi Arabia. Persons on long use of antibiotics due to chronic health problems were excluded. Data were collected over a period of four months based on a pre-structured electronic questionnaire. The questionnaire was constructed by researchers with the help of experts and intensive literature review. Results: A total of 843 participants completed the study questionnaire. Participants' ages ranged from 18 to 66 years old with mean age of 34.3 +/- 11.3 years old. The majority of participants were females (70.5%; 594) and Saudis (99.3%; 837). Exactly 796 (94.4%) of the respondents reported that Antibiotics should be purchased according to a doctor's prescription, and 450 (89%) correctly said that Antibiotics have side effects, and sometimes are dangerous. Regarding participants' attitude, 812 (96.3%) of the participants agreed that it is necessary to have additional information regarding the use of antibiotics, 688 (81.6%) agreed that the effectiveness of treatment diminishes if the full course of antibiotic therapy is not completed. As for practice, exactly 94.4% of the participants reported that they carefully read the instructions included before using antibiotics. Conclusions & recommendations: In conclusion, the study revealed that the population in Aseer region had poor knowledge regarding antibiotic use in total. Also, participants' practice was questionable and needed to be improved to adhere to safe practices with antibiotic use.