In this study, a facile method to synthesize non-toxic copper sulfide nanoparticles was reported, and the antibacterial activity of the nanoparticles in infected zebrafish was evaluated. Transmission electron microscopy showed that the CuS NPs are spherical in shape with a size range from 10 to 25 nm. FTIR spectra revealed that the CuS NPs were stabilized by N-lauryltyramine. CuS NPs exhibit very good antibacterial activity against Gram-negative and Gram-positive bacteria, and the minimum inhibitory concentration was determined as 12.5 μM. In vitro mechanistic studies revealed that CuS NPs kill the bacteria by damaging their cell membranes and producing reactive oxygen species. Therapeutic activity of CuS NPs was tested in bacteria-infected zebrafish. The route of administration of CuS NPs was via an injection and medicated bath. Both routes of drug administration were suitable to cure the fish from bacterial infection. Bacterial colony count assay revealed that the CuS NPs depleted the infectious bacteria from the fish body within 24 h. Strikingly, CuS NPs in concentration eight times than the MIC did not show toxicity in the liver and brain of zebrafish. Moreover, the NPs exhibited good hemocompatibility with human red blood cells. Our study demonstrated for the first time that CuS NPs are a safe antibacterial agent, and medicated bath with zebrafish is a hassle-free model for testing the antibacterial activity of nanoparticles.