Abstract Objective Antimicrobial stewardship programs are of paramount importance in containing antibiotic resistance and combat the acquisition of multi-drug resistance strains. Healthcare practitioners, specifically physicians and clinical pharmacists are the direct clinical antibiotic prescribing authorities. Therefore, this study aimed to assess Jordanian practitioners' perceptions and practices towards antimicrobial stewardship programs. Methods This was a cross-sectional study, in which physicians and clinical pharmacists were approached to fill out a previously validated study instrument. A close-ended structured questionnaire comprising 34 questions covering aspects related to antimicrobial stewardship program perceptions, practices and participants' demographic data were used to collect pertinent information. Key findings A total of 286 participants were enrolled in the study. There was an overall positive perception towards antimicrobial stewardship programs, while practices related to this element was still suboptimal. Additional comparison of the overall perception scores among different demographic characteristics showed that long years of practice, postgraduate studies and practice in academic sectors yielded higher perception scores (P = 0.0335, 0.0328 and 0.0007, respectively). Conclusion There is an imminent need to enhance Jordanian practitioners' knowledge about antimicrobial stewardship programs. Clear recognition of integral antimicrobial stewardship programs' components must be coupled with highlight changes in current practices related to antimicrobials usage and prescription.