Antibiotics are considered to be among the most commonly sold drug classes in Palestine. Resistance to antibiotics has increased for reasons relating to the use and misuse of antibiotics. The aim of this study was to evaluate the knowledge, and attitudes regarding antibiotic use and awareness about resistance among adults visiting the emergency departments at hospitals in North Palestine. A self-administered cross-sectional questionnaire survey involving participants aged 18 or over was conducted from June 2012 to February 2013. Adults who visited the emergency departments at hospitals in North Palestine were included. Demographic characteristics, knowledge and attitudes towards antibiotic use were included in the questionnaire. Poor and good knowledge were defined as a total knowledge score of 0–7 and 8–15 of 15 questions, respectively. Attitude scores of 0–3 and 4–7 of 7 questions were considered poor and good, respectively. A total of 375 questionnaires were included in the study. A response rate of 83.3% was attained. About 55.0% of the participants had a good knowledge and 56.5% had a good attitude towards rational antibiotic use. A significant positive correlation was shown between participants’ knowledge scores and participants’ attitude scores towards antibiotic use (R = 0.344, P = 0.001. Participants with a high family income were more likely to be aware of appropriate antibiotic use than participants with a low family income (P-value <0.001). Participants with a higher educational level (university) had a good attitude towards rational antibiotic use than those with a lower education level (P-value <0.001). This study has documented important knowledge and attitude gaps in antibiotic use. These findings will help health policymakers in Palestine to implement intervention programmes to rationalize antibiotic use. Continuing medical education, professional development and training workshops for healthcare professionals regarding rational use of antibiotics and health risks associated with the spread of antibiotic resistance are needed. In addition, minimizing non-prescription use of antibiotics and increasing the public awareness about the health and economic hazards of antibiotic resistance are also required.