This study investigated the preparation and characterization of Natamycin-incorporated agar film and its antimicrobial effect on preserve quality of fresh strawberries. Natamycin-incorporated agar films were prepared by solvent casting method and characterized in mechanical, optical, water swelling, and barrier properties. Antimicrobial activity of the films and their preservative effects on strawberries stored at 4 °C and 20 °C were also assessed. As natamycin amount increased (0%, 0.33%, 0.66%, 0.99%, 1.33%, w/w of dry film), the antimicrobial activity of the film increased with inhibition zone of Aspergillus niger and Saccharomyces cerevisiae from 0 cm to approximate 28 cm, while there was no significant effect on water vapor permeability and swelling degree. On the other hand, with the increase of natamycin, tensile strength of the film declined (from 17.61 to 14.94 MPa), while oxygen permeability enhanced from 0.161 to 0.585 × 10−15 cm3 cm/cm2 s Pa. Furthermore, the natamycin can be released quickly from antimicrobial films within 2 days under aqueous solution in accordance with fick’s law. Along with the addition of natamycin, light transmittance of antimicrobial films trends to decrease whereas the haze and total color difference of film increase significantly. Compared to control group, fungal colony number of strawberry packaged in antimicrobial film was reduced by 0.51 and 1.05 log cfu g−1 at 4 °C and 20 °C, respectively. The strawberries packaged by antimicrobial film retained high total soluble solid content with higher hardness of 61.89% and 54.47% at 4 °C and 20 °C, respectively at the end of storage. Moreover, strawberries packaged with the natamycin-incorporated film showed lower rate of water loss during storage (1.43% and 7.66% at 4 °C and 20 °C, respectively). The obtained results indicate that natamycin-incorporated agar film could be used as environment-friendly packaging alternative to preserve perishable fresh produce and extend their shelf lives by means of antimicrobial activity and other merits.