The pregnant cow urine (PCU) is an active source of antimicrobial agents that is used for fabricating chitosan coated Ag/AgCl nanoparticles (NPs) in the present study. These PCU@C-Ag/AgCl NPs were physicochemically characterized and evaluated for antimicrobial activity against selected respiratory tract infection (RTI) pathogens. The absorption band around 420 nm in UV-Visible spectrum indicated the presence of Ag NPs. The spherical shape of NPs was observed using TEM. Also, the crystalline structure was confirmed using the XRD pattern. The PCU@C-Ag/AgCl NPs showed strong antimicrobial activity against all tested RTI pathogens. In addition, FESEM analysis showed morphological changes in RTI bacterial pathogens. Thereby, PCU@C-Ag/AgCl NPs may be used as an antimicrobial material to treat RTIs in near future at clinical level.