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The unique structures and multifunctionalities of two-dimensional (2D) nanomaterials, such as graphene, have aroused increasing interest in the construction of novel scaffolds for biomedical applications due to their biocompatible and antimicrobial abilities. These two-dimensional materials possess certain common features, such as high surface areas, low cytotoxicities, and higher antimicrobial activities. Designing suitable nanocomposites could reasonably improve therapeutics and reduce their adverse effects, both medically and environmentally. In this study, we synthesized a biocompatible nanocomposite polyhydroxyalkanoate, chitosan, and tungsten disulfide (PHA/Ch-WS₂). The nanocomposite PHA/Ch-WS₂ was characterized by FESEM, elemental mapping, FTIR, and TGA. The objective of this work was to investigate the antimicrobial activity of PHA/Ch-WS₂ nanocomposites through the time–kill method against the multi-drug-resistant model organisms <i>Escherichia coli</i> (<i>E. coli</i>) K1 and methicillin-resistant <i>Staphylococcus aureus</i> (MRSA). Further, we aimed to evaluate the cytotoxicity of the PHA/Ch-WS₂ nanocomposite using HaCaT cell lines by using a lactate dehydrogenase (LDH) assay. The results demonstrated very significant bactericidal effects of the PHA/Ch-WS₂ nanocomposite, and thus, we hypothesize that the nanocomposite would feasibly suit biomedical and sanitizing applications without causing any adverse hazard to the environment.