Antimicrobial resistance (AMR) is a serious healthcare problem that affects public health globally. Appropriate understanding and knowledge of prudent antimicrobial use and resistance, along with providing evidence-based information sources, are needed for informed antibiotic prescribing practices. This study aimed to assess the knowledge, opportunity, motivation, behavior of pharmacists and their information sources regarding antibiotic use and resistance in Jordan. An online cross-sectional questionnaire was developed and administered to pharmacists during the period of July–September 2021. The survey is an adapted version of the validated European Centre for Disease Prevention and Control (ECDC) survey for antibiotic use and resistance. Pharmacists from all sectors (n = 384), of whom 276 (71.9%) were community pharmacists, completed an online questionnaire. While respondents scored highly (>87%) on knowledge on effective use, unnecessary use, and associated side effects of antibiotics, lower scores were recorded for knowledge on the spread of antibiotic resistance (52.9%). Pharmacists support easy access to guidelines on managing infections in 56% of cases, and easy access to materials advising prudent antibiotic use and resistance in 39.8% of cases. One-third of respondents (37.0%) reported no knowledge of any initiatives on antibiotic awareness and resistance. Pharmacists were aware (13.3%), unaware (36.2%), or unsure (50.5%) of the existence of a national antibiotic resistance action plan. Pharmacists indicated an interest in receiving more information on resistance to antibiotics (55.2%), medical conditions for which antibiotics are used (53.1%), how to use antibiotics (45.1%), prescribing of antibiotics (34.4%), and links between the health of humans, animals, and the environment (28.6%). Findings can inform antimicrobial stewardship with required interventions to improve antibiotic use.