Background Conducting polymer based nanocomposites are known to be effective against pathogens. Herein, we report the antimicrobial properties of multifunctional polypyrrole-cobalt oxide-silver nanocomposite (PPy-Co3O4-AgNPs) for the first time. Antibacterial activities were tested against multi-drug-resistant Gram-negative Escherichia coli (E. coli) and Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) bacteria, while antiamoebic effects were assessed against opportunistic protist Acanthamoeba castellanii (A. castellanii). Results The ternary nanocomposite containing conducting polymer polypyrrole, cobalt oxide, and silver nanoparticles showed potent antimicrobial effects against these pathogens. The antibacterial assay showed that PPy-Co3O4-AgNPs exhibited significant bactericidal activity against neuropathogenic E. coli K1 at only 8 mu g/mL as compared to individual components of the nanocomposite, whereas a 70 % inhibition of A. castellanii viability was observed at 50 mu g/mL. Moreover, PPy-Co3O4-AgNPs were found to have minimal cytotoxicity against human keratinocytes HaCaT cells in vitro even at higher concentration (50 mu g/mL), and also reduced the microbes-mediated cytopathogenicity against host cells. Conclusion These results demonstrate that PPy-Co3O4-AgNPs hold promise in the development of novel antimicrobial nanomaterials for biomedical applications.