No prior research has investigated whether the cold plasma treatment (CPT) resulted in the formation of toxic compounds. Therefore, this study carried out the experiment to check the safety of edible films treated with cold plasma by examining their acute and subacute oral toxicity in a rat model. Single-dose acute (5000 mg/kg body weight) and 14-d subacute (1000 mg/kg body weight/day) oral toxicity of cold plasma-treated edible films was assessed for male and female Sprague–Dawley (SD) rats. Rats administered 5000 mg/kg of edible film did not show the signs of acute toxicity or death after 14 d of observation. Similarly, no signs of acute toxicity or death were recorded during 14 d in rats administered 1000 mg/kg/day of edible film treated with cold plasma. Although changes in the levels of several blood components (hematocrit, hemoglobin, bilirubin, creatinine, and aspartate aminotransferase) of samples were observed, the changes compared to the control were considered to be toxicologically irrelevant as their levels were within normal physiological ranges. Macroscopic analysis showed there were no changes in color or texture of representative liver sections of SD rats following the oral administration of edible films with CPT (F-CP) or without CPT (F-NT). The results demonstrate that the cold plasma-treated edible film possessed very low toxicity, suggesting that CPT does not generate harmful by-products in the edible film.