The COVID-19 pandemic has accelerated global antibiotic usage, contributing to the rise of antimicrobial-resistant pathogens. Fish, as key players in aquatic ecosystems, have evolved unique defense mechanisms, including the secretion of antimicrobial compounds in their epidermal mucus, these antimicrobials could be used to treat antimicrobial-resistant pathogens. This study investigates the antimicrobial potential of acidic extracts from the epidermal mucus of Clarias batrachus against clinically significant pathogens. The extract demonstrated significant inhibitory effects against seven selected human pathogenic and opportunistic microbes. The antimicrobial mechanism was explored using field emission scanning electron microscopy (FESEM), revealing structural damage to the microbial cells. The physicochemical stability of the mucus compounds was experimentally validated under various conditions. Protein characterization through SDS-PAGE identified prominent bands at 11 kDa, corresponding to hemoglobin subunit-like chains (α and β), as confirmed by LC-MS/MS analysis. Bioinformatic evaluations suggested that these peptides possess not only antimicrobial but also potential antiviral and anticancer activities. Molecular docking studies further supported the applicability of these peptides against antibiotic-resistant targets (erm proteins), including NDM superbugs, highlighting their potential as novel therapeutic agents. This research underlines the promise of fish mucus-derived compounds in combating antimicrobial resistance, offering a natural and sustainable alternative to conventional antibiotics for both fish and human pathogens.