The increasing threat of bacterial infections and the limitations of conventional antibiotics have intensified the search for innovative antimicrobial substances. This study examines a heterostructure nanomaterial of single layer graphene (SLG) and delaminated MXene (d-Ti3C2Tx), designed to efficiently inhibit bacterial growth. MXene was synthesised using selective etching and delamination, while the SLG/d-Ti3C2Tx composite was prepared via ultrasonication to ensure uniform dispersion and interfacial interaction between the materials. Powder X-ray diffraction (PXRD), FTIR, and FE-SEM confirmed the successful integration of the 2D d-Ti3C2Tx and SLG. Antibacterial activity was assessed using two methods: optical density and colony-forming unit (CFU) quantification. At 500 µg/mL, the SLG/d-Ti3C2Tx heterostructure demonstrated the strongest antibacterial activity among all materials investigated. Low CFU counts and significant inhibition of bacterial growth were observed. The enhanced activity is attributed to the large surface area of graphene and the sharp edges and surface functionalities of d-Ti3C2Tx, which damage microbial membranes and obstruct cellular processes. The results clearly demonstrate that SLG/d-Ti3C2Tx acts as an effective antibacterial agent. This study opens new avenues for the future development of 2D heterostructures engineered for microbial resistance under diverse conditions. Thus, the designing of the 2D/2D heterostructure SLG/d-Ti3C2Tx is a promising strategy to achieve the antimicrobial activity for various applications.