Zoonotic infections caused by bacterial pathogens are considered as major threat to humans and the aquaculture industry. This problem triggered the search for various natural products from plants, microorganisms, animal tissues, and secretions to determine the presence of metabolites that may be of potential antimicrobial effects against infectious agents. However, limited attempts have been conducted to elucidate the potential use of freshwater fish mucus in against pathogens. Here, the antimicrobial activity of mucus of economically-important freshwater fish species in the Philippines: Oreochromis niloticus (tilapia), Clarias batrachus (catfish), and Channa striata (snakehead fish) was investigated against fish and human pathogens. The pooled fish mucus was extracted with succeeding centrifugation and filtration. The acidic mucus extracts were tested for antimicrobial-inhibitory effects and minimum inhibitory concentration (MIC) by agar-overlay diffusion and microbroth plate dilution method, respectively. The results showed that all fish mucus extracts exhibited antimicrobial effects against test pathogens with catfish exhibiting the highest inhibitory effects against Pseudomonas aeruginosa (p = 0.096), Klebsiella pneumoniae (p = 0.000), Enterococcus faecalis (p = 0.665), Micrococcus luteus (p = 0.000), Aeromonas hydrophila (p = 0.000), Staphylococcus aureus (p = 0.000), Escherichia coli (p = 0.000), and Serratia marcescens (p = 0.000) as compared to the broad-spectrum antibiotic control, Cefoperazone. Interestingly, catfish mucus revealed inhibitory effects against Gram-positive S. aureus and M. luteus at the lowest concentration (1:4 dilution). The present findings revealed the potential antimicrobial use of freshwater fish mucus against medically-important pathogens.