Since graphene's exodus, layered materials’ exfoliation to two-dimensional (2D) nanosheets has attracted much attention. The primary layered materials are composed of weak van der Waals interlayer forces and strong intralayer bonds. Biotite (BTT) is one of the members of mica clays, which has stronger interlayer forces. In this study, three various procedures have been reported to exfoliate BTT. In the first method, ultrasound waves weakened the interlayer forces and so caused the exfoliation of the BTT to 2D nanosheets. In the second route, thermal and facile exfoliation of BTT intercalated compounds were combined. In the third one, a thermal exfoliation method was performed. These improved synthesis methods for producing few-layered exfoliated BTT (BTN) nanosheets, which are cost-effective, reproducible, and scalable, obtained BTNs with thicknesses of 10–50 nm, while the specific surface areas were increased by 4 times. The high specific surface area and sharp edges of BTN provide higher potentials for the decomposition of Escherichia coli and Staphylococcus aureus cells. The bactericidal effect of the BTNs against different bacteria was evaluated through the colony-forming unit (CFU) procedure and the minimum inhibitory concentration (MIC) quantities was subsequently measured. According to the results, after exfoliation, the antibacterial performance was improved about 8 times.