In this study, two types of antibacterial montmorillonites (Mt) were prepared using a facile method. The Mt modified with ε-polylysine hydrochloride (ε-PL) was named PL-Mt, while the Mt dually modified with dioctadecyl dimethylammonium chloride (D1821) and ε-PL was named PL-OMt. The results of the X-ray diffraction, Fourier-transform infrared (FTIR) spectroscopy and thermogravimetric analysis (TGA) of the PL-Mt indicated that 30% ε-PL was the most suitable amount for intercalating the Mt. The particle size and distribution of the ε-PL in the solution demonstrated that the Mt d-value could not be further increased owing to the increasing ε-PL diameter. The result of the X-ray diffraction of PL-OMt displayed that ultrasonic treatment at 600 W facilitated ε-PL to intercalate into the OMt interlayer space. The PL-OMt prepared with ultrasonic treatment at 600 W exhibited antibacterial activity against Escherichia coli and Bacillus subtilis superior to that of the PL-OMt prepared with higher-power ultrasonic treatment. Thus, the addition of 30% ε-PL based on the dry Mt mass is the most suitable ratio for preparing PL-Mt, while ultrasonic treatment at 600 W is the most suitable for preparing PL-OMt. These findings may expand the application fields of ε-PL.