Reactive oxygen species (ROS) are ideal alternative antibacterial reagents for rapid and effective sterilization. Although a variety of ROS-based antimicrobial strategies have been developed, many are still limited by their inefficiency. Herein, we report the synthesis of the Ag-MXene nanozyme, which have superior peroxidase-like activity for antibacterial applications. As a result, Ag-MXene nanozyme can efficiently increase the level of intracellular ROS, converting H₂O₂ into hydroxyl radicals that effectively kill both Gram-negative and Gram-positive bacteria and disrupting the bacterial biofilm formation. Moreover, a sensitive and selective colorimetric biosensor was constructed for assaying cholesterol based on the Ag-MXene’s prominent peroxidase-mimicking activity and the cholesterol oxidase cascade reaction. The biosensor exhibits high performance with a linear cholesterol detection range of 2–800 μM, and a detection limit of 0.6 μM. Ag-MXene nanozyme can be used for the rapid detection of cholesterol in serum without complicated sample pretreatment. Collectively, it is conceivable that the proposed Ag-MXene nanozyme could be used as a biocide and as a cholesterol sensor. This study provides a broad prospect for the rapid detection and sterilization of MXene nanozymes in the biomedical field.