The characteristics of two-dimensional (2D) nanomaterials have attracted remarkable attention, particularly in the fields of environmental engineering, sciences and medicine. This study was focused on Ti3C2Tx-MXene, a novel 2D nanomaterial known for its antibacterial efficacy against both Gram-negative and Gram-positive bacteria. Our investigation has represented the initial exploration of the primary antifungal capabilities of Ti3C2Tx-MXene nanosheets, with variations in lateral size induced by ultrasonication time. The Ti3C2-MXene, characterized by negative surface potential, was tested against Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia, Enterobacter cloacae and fungal strains of Candida albicans. Ultrasonication was employed without the use of any chemical intercalant for delamination, separation, and exfoliation. The optimal ultrasonication duration was determined, revealing the minimum inhibitory concentrations of 8 μl/mL and 4 μl/mL against E. coli and S. aureus, respectively, as observed through the broth dilution test. The antifungal activity against Candida albicans has been assessed using the well diffusion method, revealing a substantial zone of inhibition measuring 14 mm. The demonstrated antimicrobial efficacy of ultrasonicated MXene sheets has shown a huge potential for serving as an effective anti-biofouling material.