The aim of this study was to investigate the effect of atmospheric cold plasma (ACP) treatment on the microbial inactivation, physicochemical properties, and shelf-life of strawberry fruit with its extended in-package storage at room (25 °C) and refrigerated (4 °C) temperature. ACP treatment of 10, 15 and 30 min was studied on strawberry fruit using a dielectric barrier discharge (DBD) at 60 kV with an input voltage of 260 V at 50 Hz. The shelf-life of ACP treated strawberry was extended to 5 days at 25 °C and 9 days at 4 °C in sealed ACP package. However, non-treated packaged strawberry was degraded in 2 days. ACP treatment of 15 min resulted in 2 log reduction of microbial load and enhanced the concentration of chlorogenic acid, hyprin, phloretin, vanillin, gallic acid, 4-hydroxybenzaldehyde and rutin during in-package storage of 5 day (~ 120 h) at 25 °C with respect to control ( < 0.05). In addition, ACP treatment of 15 min at 60 kV was also found to increase the total phenolic content and antioxidant activity. However, total soluble solids, pH and moisture were not affected with ACP treatment ( > 0.05). Therefore, ACP treatment of 15 min with in-package storage of 5 days (~ 120 h) was found to be advantageous for increasing the shelf-life and functional quality of strawberry fruit.