Multi-layer (ML) Ti3C2Tx was investigated regarding its anti-extended spectrum beta-lactamases (ESBLs) activity and in-vivo toxicity. Powder X-ray diffraction of ML-Ti3C2Tx confirmed the successful etching of Ti3AlC2 (MAX) to form ML-Ti3C2Tx as well as the presence of -OH,-F and-O surface terminations. Fourier-transform infrared spectroscopy and thermal decomposition verified the presence of its typical chemical bonds and moieties including Ti-O, O-H, C-H, C--O, and -OH, which further confirmed the successful synthesis of ML-Ti3C2Tx. Scanning and transmission electron microscopy demonstrated its multi-layered structure, while X-ray photoelectron spectroscopy revealed the presence of various photoelectron peaks with binding energy positions corresponding to O1s, C1s, Ti 2p, and F1s, indicating the existence of distinct surface terminations and oxidation states. The E-strip synergy and micro-iodometric methods suggested that ML-Ti3C2Tx possesses excellent beta-lactamases inactivation with 96 and 94 % inhibition at 90 mu g/mL against P. aeruginosa (MN310553) and K. pneumoniae (MN368594), respectively. Concentration-dependent reactive oxygen species (ROS) generation of ML-Ti3C2Tx decreased notably bacterial cell growth as well as its response to beta-lactamase resistances. Moreover, intercellular damage and outer cell wall rupture through external proliferation forces of sharp-edged ML-Ti3C2T were confirmed by confocal laser and scanning electron microscopy. Molecular docking suggested that Ti3C2Tx induced effective inhibition of TEM1 beta-lactamase, providing insights for future development of drug-resistant ESBLs with gene control. Finally, the evaluation of the in-vivo toxicity of the synthesized ML-Ti3C2Tx against brine shrimp Artemia revealed their non-toxicity up to a maximum concentration of 150 mu g/mL and 24 h exposure time.