Abstract Despite intensive research on the application of two-dimensional (2D) materials, including MXenes, in nanomedicine, the knowledge concerning the mechanisms responsible for their observed bio-effects is far from being understood. Here we present insight into the mechanism of toxicity in vitro of the 2D Ti₃C₂ MXene. The most important results of this work are that using simple, inexpensive, post-delamination treatments, such as ultrasonication or mild thermal oxidation it is possible to ‘tune’ the cytotoxicity of the Ti₃C₂T _z flakes. Sonication of Ti₃C₂T _z flakes, or sonication followed by mild oxidation in the water at 60 °C, renders them selectively toxic to cancer cells as compared to non-malignant ones. It relates to the appearance of superficial titanium (III) oxide (Ti₂O₃) layer corresponding to the type of post-treatment. The presence of surface-Ti₂O₃ results in a noticeably higher generation of oxidative stress compared to pristine 2D Ti₃C₂. Our findings give evidence that the sonication and thermal treatments were successful in changing the nature of the surface terminations on the Ti₃C₂T _z surfaces. This study makes a significant contribution to the future rationalized surface-management of 2D Ti₃C₂ MXene as well as encourages new rationalized applications in biotechnology and nanomedicine. Bullet points: 1. First study on 2D Ti₃C₂ MXene superficially oxidized to titanium (III) oxide i.e. Ti₂O₃. 2. By sonication Ti₃C₂Tz MXene flakes followed by mild thermal oxidation in the water at 60 °C for 24 h, it is possible to ‘tune’ the toxicity of the flakes to cancerous cell lines. 3. Decreases in cell viabilities were dose-dependent. 4. Highest cytotoxic effect was observed for thermally oxidized samples. 5. The thermally oxidized samples were also selectively toxic towards all cancerous cell lines up to 375 mg l⁻¹. 6. Reactive oxygen species generation was identified as a mechanism of toxicity.