Biopolymer-based packaging films possess outstanding performances and are being developed as the alternatives to traditional petroleum-based plastic packaging films with many non-ignorable shortcomings. In this study, chitosan, succinylated pullulan (SP), and berberine (BBR) were combined to fabricate novel biopolymer-based composite films (CSSPB) via the layer-by-layer assembly method. The effects of the incorporation of BBR on the physicochemical properties of the film were investigated. It was found that after BBR was added, the tensile strength (TS), elongation at break (EAB), hydrophobicity, and antioxidant capacities of the film were enhanced. The chemical bonding, crystalline properties, elemental composition, and thermal stability of the films were also characterized by Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and thermogravimetric analysis (TGA), respectively. The in vitro antifungal tests revealed the antifungal activities of the films with a relatively high BBR content against Colletotrichum gloeosporioides (CG). In the preservation experiments, the CSSPB films exhibited preservation effects on fresh-cut apples, which manifested as delaying browning, weight loss, an increase in the soluble solids content, and a decrease in hardness. The new CSSPB composite films were opined to hold application potential in the field of food packaging.