Strategies to overcome the limitations of konjac glucomannan (KGM) films, including their poor mechanical properties, low water resistance, and limited bioactivity, are being developed. In this study, fucoidan-chitosan nanoparticles encapsulating tea polyphenols (TP-FCNPs) were prepared using an electrostatic self-assembly method. Subsequently, a novel bio-nanocomposite film was prepared by incorporating TP-FCNPs as fillers into a KGM-based membrane. The microstructural, physical, and biological properties of the nanocomposite films were evaluated. Scanning electron microscopy (SEM) observations showed that in the KGM/TP-FCNPs containing 1–7% TP-FCNPs, the TP-FCNPs were uniformly distributed throughout the KGM film matrix. The highest tensile strength of KGM/TP-FCNPs films was 33.05 ± 1.57 MPa, which was 404.53% that of pure KGM film. Incorporating TP-FCNPs also significantly enhanced the UV absorption ability and antimicrobial and antioxidant activities of the bio-nanocomposite films, while reducing their transmittance. In addition, KGM/TP-FCNPs used as packaging effectively reduced fruit mass loss and firmness loss, inhibited microbial growth, and significantly prolonged fruit shelf-life on day 21 of the cherry tomato (Lycopersicon esculentum var. cerasiforme A. Gray) packaging trial. In conclusion, the KGM/TP-FCNPs bio-nanocomposite films exhibit satisfactory properties, and thus, show potential for applications in fruit preservation.