Abstract The kidney bean protein isolate–chitosan (KPI–CH) composite films were fabricated by using ultrasonic pretreatment to produce flexible films as the carriers for antimicrobial nisin. Tensile and thermal properties, as well as opacity of the composite films were evaluated. The surface and transversal microstructure of the composite films was investigated by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), and an attempt was made to relate tensile properties of the films to their microstructure attributes. The complex between KPI and CH with ultrasonic pretreatment produced ductile films, and elongation at break (EAB) of the films increased to about 76.57% upon increasing CH-to-KPI ratio. The near-surface of the films changed gradually from protein continuous to bi-continuous, and CH continuous pattern upon increasing CH-to-KPI ratios, whereas the core of the films exhibited a different pattern. The compositional variety appeared between the near-surface and the core of the films, whereas the core of the films did not show an apparent compositional gradient, exhibiting bi- or CH-continuous patterns in a CH-to-KPI ratio dependent manner. Microscopical structure for transversal sections and tensile properties of the films strongly indicated that KPI and CH were partially compatible within film matrices. Nisin loading endow KPI films with antimicrobial activity against Bacillus subtilis and Staphylococcus aureus, especially the films produced at pH 3.0, and enhanced antimicrobial activity of the composite films. The results presented here supported that the nisin-loaded KPI–CH blend films are expected to serve as antimicrobial food-packaging.