More research is needed on the drivers of irrational antibiotic prescribing among healthcare professionals and to ensure effective prescribing and an adequate understanding of the issue of antibiotic resistance. This study aimed at evaluating prescribers’ knowledge, attitudes and behaviors about antibiotic use and antibiotic resistance. A cross-sectional study was conducted utilizing an online questionnaire and included physicians and dentists from all sectors in Jordan. A total of 613 prescribers were included (physicians n = 409, dentists n = 204). Respondents’ knowledge on effective use, unnecessary use or associated side effects of antibiotics was high (>90%), compared with their knowledge on the spread of antibiotic resistance (62.2%). For ease of access to the required guidelines on managing infections, and to materials that advise on prudent antibiotic use and antibiotic resistance, prescribers agreed in 62% and 46.1% of cases, respectively. 28.4% of respondents had prescribed antibiotics when they would have preferred not to do so more than once a day or more than once a week. Among respondents who prescribed antibiotics, 63.4% would never or rarely give out resources on prudent use of antibiotics for infections. The findings are of importance to inform antibiotic stewardships about relevant interventions aimed at changing prescribers’ behaviors and improving antibiotic prescribing practices.