A set of chitosan/ε-polylysine (ε-PL) bionanocomposite films were prepared by a simple in situ self-assembly technique using sodium tripolyphosphate (TPP) as cross-linking agent. The physical, mechanical, structural, and antimicrobial properties of these films were investigated. Fourier infrared spectroscopy and X-ray diffraction showed that the introduction of TPP promoted the formation of hydrogen bonds and electrostatic interactions among functional groups of chitosan or ε-PL, which improved the tensile strength and decreased the water solubility, water vapor permeability and surface wettability of films. On the other hand, the incorporation of ε-PL weakened the bionanocomposite film' structure and integrity, resulting in a decrease trend of films' mechanical and barrier properties. More importantly, the bionanocomposite films exhibited excellent antimicrobial efficacy against E. coli and S. aureus by the increasing ratio of ε-PL. And ε-PL presented a sustained release from the films, which was closely related to TPP concentration. Results of this study suggested that chitosan/ε-PL films could be used as antimicrobial bio-material and have great potential in food industry.