The aim of this study is to evaluate the knowledge and behavior toward antibiotic self-medication among medical and non-medical university students in Iran. A cross-sectional study was conducted on a sample of 200 students randomly chosen from a medical and a non-medical university in Ahwaz, South of Iran in 2008. Data was collected using self administered questionnaires with open-ended and close-ended items. Data were entered and analyzed using SPSS 14 and the results were presented as the percentage 97.5% of respondents filled and returned the questionnaire. Self-medication with antibiotics was reported by 42.2% of the medical and 48% of the non-medical students during the last 3 months. Respiratory problems such as sore throat and common cold was the main indication for self-medication with antibiotics (73.3%) and amoxicillin was the most commonly used antibiotic in both groups. The choice of self-medication was based on medical knowledge among medical students (50%) and on a previous suggestion by a physician (32.6%) for the non-medical ones. The prevalence of self-medication with antibiotics among medical and non-medical students was high. Educational programs are needed to teach university students about the potential problems of self-medication with antibiotics.