The emergence of bacterial resistance is increasing continuously due to the misuse of antibiotics that results in difficulty controlling the bacterial infection or the lack of treatment. Therefore, a new class of antibiotic or nano-antibiotic materials must be developed to treat bacterial infectious diseases. In this context, the present study focuses on synthesizing Cu-metal incorporated WS2 nanosheet (Cu-WS2-NS)-based antibiotic materials. The Cu-WS2-NS was synthesized via a liquid exfoliation process, followed by the incorporation of Cu-metal using a microwave process. The as-prepared WS2-NS and Cu-WS2-NS based antibiotics were characterized via SEM, EDS, and FT-IR analysis. The as-prepared WS2-NS and Cu-WS2-NS based antibiotic materials showed high biocompatibility or insignificant toxicity (similar to 0.78% and 3.7%) against erythrocyte cells, respectively. The as-prepared WS2-NS and Cu-WS2-NS based antibiotic materials effectively inhibited or killed (99.99%) both E. Coli and S. aureus bacterial strains. The antibacterial activity of the Cu-WS2-NS based antibiotic materials was mainly due to the Cu metal that binds with the sulfide group of WS2-NS. Interestingly, the Cu-WS2-NS-based antibiotic materials showed superior or comparable antibiotic ability to that of commercially available Ampicillin antibiotics. Therefore, the as-prepared Cu-WS2-NS based antibiotic materials might be potential emerging materials to treat infectious diseases or control bacterial infections.