Neem leaves extract was incorporated into the matrix of seaweed biopolymer, and the seaweed-neem biocomposite films were irradiated with various doses of gamma irradiation (0.5, 1.5, 2.5, 3.5, and 4.5 kGy). The physical, barrier, antimicrobial, and mechanical properties of the films were studied. The incorporation of 5% w/w neem leaves extract into a seaweed-based film, and gamma irradiation dose of 2.5 kGy was most effective for improved properties of the film. The results showed that the interfacial interaction of the seaweed-neem improved with physical changes in colour and opacity. The water solubility, moisture content, and water vapour permeability and biodegradability rate of the film reduced. The contact angle values increased, which was interpreted as improved hydrophobicity. The tensile strength and modulus of the films increased, while the elongation of the composite films decreased compared to the control film. The film’s antimicrobial activities against bacteria were improved. Thus, neem leaves extract in combination with the application of gamma irradiation enhanced the performance properties of the film that has potential as packaging material.