Rubber seed shell (Hevea brasiliensis) was used as a low-cost substrate to produce lactic acid via Lactobacillus plantarum LB2. The medium components were initially screened by two-level full factorial design. Three variables (pH, moisture, and polyoxyethylene sorbitan monooleate (PSM)) were used in the central composite design and response surface methodology. The amount of PSM was found to be a significant variable in lactic acid production. Lactic acid was purified and used for the chemical fabrication of a polylactic acid-chitosan composite film. Compared with the polylactic acid, the composite film improved the tensile strength, elongation strength, and tearing strength. The film prepared with 1% chitosan-polylactic acid exhibited the maximum antibacterial activity against Bacillus cereus (21 ± 1 mm) and the lowest activity against Escherichia coli (10 ± 1 mm). The polylactic acid-chitosan film prepared with 1% chitosan was used as a packing material to store the fish fillets and presented reduced mesophilic (4.3 ± 0.1 Log CFU/g) and psychrotropic (3.2 ± 0.2 Log CFU/g) bacterial populations compared with those of the control (4.9 ± 0.2 Log CFU/g and 3.7 Log CFU/g). Rubber seed shells can be used as an alternative culture medium for lactic acid production, to reduce the production cost of polylactic acid.