Investigating new antimicrobial agents from various biological sources is necessary due to the rise of bacteria that are resistant to drugs. A neglected source of bioactive substances with possible antibacterial qualities is fish, especially Oreochromis niloticus (Nile tilapia). This study identifies the bioactive components of extracts made from O. niloticus and examines their antibacterial activity. PBS and sterile water were used to remove fish mucus using a solvent. Using the agar well diffusion method, extracts were tested for antibacterial activity against a panel of Gram-positive bacteria (Staphylococcus aureus, B. cereus), Gram-negative bacteria (Klebsiella pneumoniae, P. aeruginosa), and fungus (Candida albicans). The presence of hydroxyl and amide functional groups, which are suggestive of proteins and polyphenolic substances, was further validated by Fourier-transform infrared spectroscopy (FTIR). The study demonstrates O. niloticus’s antibacterial potential, with its mucus being an exceptionally abundant source of bioactive substances. These results highlight the potential of fish-derived antimicrobial compounds as substitutes for traditional antibiotics. The isolation, structural clarification, and possible therapeutic uses of these chemicals should be the main areas of future study. Furthermore, to guarantee the efficacy and safety of these natural compounds, knowledge of their toxicity profiles and mechanisms of action is crucial. In the fight against antibiotic resistance, this study helps to establish sustainable bioresources and expands our knowledge of O. niloticus’s antibacterial potential.