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With the demand for better chronic wound care, the clinical problem of managing wound infections has already reached a critical level. Therefore, the development of novel antibiotic-free wound dressings is essential for wound care. The proteolytic enzyme bromelain, silver nanoparticles (AgNPs), and bromelain loaded on silver nanoparticles (Br-AgNPs) were assessed for their impact on wound healing in the albino mice. The formulated nanoparticles were characterized using different techniques, i.e., ultraviolet (UV), Fourier transform infrared (FTIR), and X-ray diffraction (XRD) analysis. The antibacterial and fungal activities of the formulated nanoparticles were also investigated. After 5 days of treatment, the wound healing progression and wound size reduction rate were monitored. In addition, serum and skin tissue samples were collected to assess the changes in inflammation levels and observe the histological changes. The results indicated a spherical shape and regular distribution of the synthesized gold nanoparticles, with an average particle size of 41.33–61.30 nm, where the size was about 39.84 and 82.91 nm after loading bromelain. The Br-AgNPs were the most active antimicrobial agents against Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Candida albicans, as compared to the other samples and positive controls. The fibronectin and collagen III levels were significantly increased in the animal groups treated with bromelain and Br-AgNPs (7132.52 ± 324.67 and 7006.00 ± 565.19 pg/ml, respectively), compared to the negative control group. Moreover, wound closure was significantly enhanced in the Br-AgNP-treated animals compared to the other treated and control groups. The histopathological analysis supported the wound healing potential of the Br-AgNPs through their ability to induce dense fibrous connective tissue in the wound area, which was rich in irregular bundles of collagen fibers and interspersed with numerous fibroblasts. The present findings could provide valuable insight into how Br-AgNPs can accelerate wound healing, reduce recovery time, and provide an antimicrobial barrier for wounds. [ABSTRACT FROM AUTHOR]