Abstract The study aimed to improve polysaccharide-based packaging to extend the shelf life of fresh produce; a composite film with antimicrobial function was developed and tested. The packaging film is a blend of agar, a film-forming substrate; carrageenan, as a reinforcing agent; glycerol, as a plasticizer; and Nisin, an antimicrobial agent. The film was evaluated for its antibacterial, mechanical, and barrier properties at different humidity levels, using fresh-cut watermelon as the test food material. The film effectively inhibited the growth of Staphylococcus aureus and Listeria monocytogenes. Under relative humidity of 20%, 50%, and 90%, the tensile strength of the antibacterial film containing 0.28% Nisin was 23.08 ± 0.65, 16.09 ± 1.73, and 6.52 ± 0.56 MPa, respectively, and the film also had excellent barrier and heat-sealing properties. The packaging test using fresh-cut watermelon sealed in the antibacterial film containing 0.28% Nisin, under controlled atmosphere storage at either 4 °C or 20 °C, effectively inhibited microbial colonization in the melon and slowed the deterioration of the fruit, as indicated by measures of hardness, weight loss, and soluble solids. This method can extend the shelf life of fresh-cut fruit and provide a reference for further research on polysaccharide-based protective film for fresh produce.