The study investigates the antibacterial properties of Ti3C2Tx MXene, a two-dimensional transition metal carbide, against both Gram-negative (E. coli) and Gram-positive (B. subtilis) bacteria. The antibacterial activity was evaluated using optical density (OD) measurements and colony-forming unit (CFU) counts. Ti3C2Tx showed higher antibacterial efficiency compared to graphene oxide (GO), with over 98% bacterial cell viability loss observed at 100 μg/mL within 4 hours. The mechanism of action was investigated using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and lactate dehydrogenase (LDH) release assays, which revealed damage to the bacterial cell membrane and cytoplasmic leakage. The antibacterial activity was concentration-dependent, and Ti3C2Tx induced oxidative stress in bacteria, as evidenced by glutathione oxidation assays. The study concludes that Ti3C2Tx is a promising 2D antimicrobial nanomaterial with potential applications in water treatment and biomedical fields.The study investigates the antibacterial properties of Ti3C2Tx MXene, a two-dimensional transition metal carbide, against both Gram-negative (E. coli) and Gram-positive (B. subtilis) bacteria. The antibacterial activity was evaluated using optical density (OD) measurements and colony-forming unit (CFU) counts. Ti3C2Tx showed higher antibacterial efficiency compared to graphene oxide (GO), with over 98% bacterial cell viability loss observed at 100 μg/mL within 4 hours. The mechanism of action was investigated using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and lactate dehydrogenase (LDH) release assays, which revealed damage to the bacterial cell membrane and cytoplasmic leakage. The antibacterial activity was concentration-dependent, and Ti3C2Tx induced oxidative stress in bacteria, as evidenced by glutathione oxidation assays. The study concludes that Ti3C2Tx is a promising 2D antimicrobial nanomaterial with potential applications in water treatment and biomedical fields.