Antimicrobial resistance (AMR) is leading to greater therapeutic cost, length of hospital stays, adverse events, morbidity and mortality. Hospital-based antimicrobial stewardship programs (ASPs) engaging physicians, pharmacists, microbiologists and nurses are considered as effective way to ensure appropriate use of antimicrobial agents. The aims of our study were to assess nurses' perception, involvement, confidence and barriers towards hospital-based ASPs, and use the findings to provide future guidance. Methods: A web-based, cross-sectional study was conducted among the nurses serving at eleven hospitals of Punjab province of Pakistan during a period of two months (December 2021-January 2022). Data were collected using a validated self-administered questionnaire. All data were analyzed using SPSS version 22. Results: A total of 583 nurses participated in the study (response rate = 77.7%). All the participants were female staff nurses and 86% had a minimum of 3 years of working experience. The overall median score on the perception towards ASPs among the nurses was 18 (IQR: 16, 19) on a 0 to 20 scale whereas median score on the involvement in ASPs was 18 (IQR: 15, 37) on a 0 to 64 scale. Median perception and involvement score were statistically significantly differed by age (p < 0.001) and years of experience (p < 0.001). The overall median score on the confidence to perform ASP activities was 22 (IQR: 14, 24) on a 0 to 28 scale. Lack of knowledge, insufficient support from administration, and heavy workload were found to be the common barriers to perform ASP. Conclusion: Our study concluded that Pakistani nurses have positive perception but limited involvement in ASPs. Moreover, they are confident to perform ASP besides many barriers.