Antimicrobial irradiated seaweed–neem biocomposite films were synthesized in this study. The storage functional properties of the films were investigated. Characterization of the prepared films was conducted using SEM, FT-IR, contact angle, and antimicrobial test. The macroscopic and microscopic including the analysis of the functional group and the gas chromatography-mass spectrometry test revealed the main active constituents present in the neem extract, which was used an essential component of the fabricated films. Neem leaves’ extracts with 5% w/w concentration were incorporated into the matrix of seaweed biopolymer and the seaweed–neem bio-composite film were irradiated with different dosages of gamma radiation (0.5, 1, 1.5, and 2 kGy). The tensile, thermal, and the antimicrobial properties of the films were studied. The results revealed that the irradiated films exhibited improved functional properties compared to the control film at 1.5 kGy radiation dosage. The tensile strength, tensile modulus, and toughness exhibited by the films increased, while the elongation of the irradiated bio-composite film decreased compared to the control film. The morphology of the irradiated films demonstrated a smoother surface compared to the control and provided surface intermolecular interaction of the neem–seaweed matrix. The film indicated an optimum storage stability under ambient conditions and demonstrated no significant changes in the visual appearance. However, an increase in the moisture content was exhibited by the film, and the hydrophobic properties was retained until nine months of the storage period. The study of the films antimicrobial activities against Staphylococcus aureus (SA), and Bacillus subtilis (BS) indicated improved resistance to bacterial activities after the incorporation of neem leaves extract and gamma irradiation. The fabricated irradiated seaweed–neem bio-composite film could be used as an excellent sustainable packaging material due to its effective storage stability.