Background: The misuse of antibiotics in low-and middle-income countries is getting escalated leading to the emergence of antimicrobial resistance. This study aims to evaluate the knowledge, behavior, and practice of antimicrobial misuse among the Pakistani population for the implementation of an effective stewardship plan that ensures the rational use of medicines. Methods: It was a nationwide cross-sectional survey conducted among 1011 participants in Pakistan from March 2022 to August 2022. Statistical analysis was done through SPSS v21. The original Bloom's cut-off point was utilized to categorize the participants' overall knowledge, behavior, and practice. The χ 2 test was used to test for group differences. For multivariable logistic regression analyses, adjusted odds ratio (AOR) was calculated at 95% CI. Results: The majority of the respondents were female 69.3%, in a state of good health 81.5%, and non-health care professionals 64.0%. More than half of the participants (55.3%) demonstrated true knowledge, whereas more than four-fifths (88.6%) and almost half (47.9%) of the participants showed moderate to good behavior and practice about the rational use of antimicrobials. The employed participants (AOR: 2.12, 95% CI: 1.51-2.98), health professionals (AOR: 1.64, 95% CI: 1.25-2.17), urban residents (AOR: 1.61, 95% CI: 1.12-2.38) and participants without comorbidities (AOR: 1.89, 95% CI: 1.32-2.70) had significant association with good/true knowledge. Lower education (AOR: 1.69, 95% CI: 1.06-2.70), rural residence (AOR: 2.06, 95% CI: 1.42-3.01), and lower monthly income <50 PKR (AOR: 1.67, 95% CI: 1.28-2.17) were significant factors affecting practice about rational use of antimicrobials. However, none of the factors had significant association with behavior of the participants. Conclusion: Implementation of an effective management plan for rational use of medicines is suggested to halt antibiotic misuse which harms public health. Adequate training programs for health care professionals and educational interventions for the general public are required. Strict enforcement of rules and regulations, Drug Sale Rules 2007, and implementation of Pakistan's National Action Plan on antimicrobial resistance are essential.