This study investigated the knowledge, attitudes and behaviours towards antibiotics among the general public living in the Republic of Cyprus (RoC) and the Turkish Republic of Northern Cyprus (TRNC) by using an online questionnaire. Differences were examined using independent samplest-tests, chi-square tests, Mann–Whitney U tests and Spearman’s rho. In total, 519 individuals completed the survey (RoC = 267, TRNC = 252), with an average age of 32.7, and 52.2% were female. Most citizens correctly identified paracetamol (TRNC = 93.7%, RoC = 53.9%) and ibuprofen (TRNC = 70.2%, RoC = 47.6%) as non-antibiotic medications. A substantial proportion thought antibiotics could treat viral infections, such as a cold (TRNC = 16.3%, RoC = 40.8%) or the flu (TRNC = 21.4%, RoC = 50.4%). Most participants understood that bacteria can become resistant to antibiotics (TRNC = 71.4%, RoC = 64.4%), that unnecessary use can lead to drug ineffectiveness (TRNC = 86.1%, RoC = 72.3%) and that they should always complete the course of antibiotics (TRNC = 85.7%, RoC = 64.0%). Positive attitudes towards antibiotics correlated negatively with knowledge in both samples, indicating that the more people know, the less positive their attitudes towards their use. The RoC appears to have tighter controls of over-the-counter (OTC) sales of antibiotics than TRNC. This study reveals that different communities can have varying levels of knowledge, attitudes and perceptions about antibiotic use. Tighter enforcement of the OTC regulations, educational efforts and media campaigns are needed for enhancing prudent antibiotic use on the island.