We aimed to assess the knowledge and understanding of antibiotic use and resistance in the general population of Cyprus, in order to inform future antibiotic awareness campaigns with local evidence. Cross-sectional survey following the methodology of the “Antibiotic resistance: Multi-country public awareness survey” of the World Health Organization, during December 2019–January 2020. A total of 614 respondents participated: 64.3% were female and most were aged 35–44 years (33.2%) or 25–34 years (31.8%). One-third had used antibiotics >1 year ago and 91.6% reported receiving advice on appropriate use from a medical professional. Despite high awareness on correct use of antibiotics, lack of knowledge was noted for specific indications, where approximately one-third believed that viral infections respond to antibiotics and 70.7% lack understanding of how antibiotic resistance develops. Higher education graduates exhibited significantly higher knowledge rates. As high as 72.3% were informed about “antibiotic resistant bacteria” from healthcare professionals or social media. Most agreed on the usefulness of most suggested actions to address antibiotic resistance, with higher proportions acknowledging the role of prescribers. Up to 47% could not identify their role in decreasing antibiotic resistance. Our study provides local evidence to inform future efforts in a country characterized by high antibiotic consumption rates.