Ti3C2TX nano-sheets (MXenes) with excellent light-conversion capacity have gained importance in treating infectious diseases due to their limited bacterial resistance. In this study, we exploit this property to design photothermal antibacterial therapy using few- (FX) and multi-layer (MX) Ti3C2Tx nano-sheets. We demonstrate that FX have a higher cytocompatibility and conversion of light to heat, but MX show a better efficacy in inhibiting growth of S. aureus and E. coli due to MXenes’ reversible bacteria trapping. For MX (25 µg/mL), □37% of E. coli and □23% of S. aureus cells survived, while the effect was less pronounced for FX with □72% of E. coli and □46% of S. aureus viable cells after treatment. After using 100 µg/mL of MX, □11% of E. coli and □4% of S. aureus survived, while FX had only a mild effect on both species. The NIR laser treatment increased the efficacy of both materials: 100 µg/mL of MX combined with 5 min laser treatment at 5.7 W cm − 2 completely killed both species. For FX, the treatment with 3 W cm − 2 and the highest concentration (100 µg/mL) induced an effect comparable to MX (87% on E. coli, 95% on S.aureus). The combined NIR-MXene treatment causes an irreversible cell death linked to the loss of cell integrity (DNA release quantification and bacteria debris observation).