Cold plasma (CP) has become an alternative to conventional thermal processing of food products. In this study, the effect of cold plasma treatment time on the inactivation and quality of tilapia fillets was investigated. The surfaces of tilapia fillets were inoculated with Salmonella enteritis (S. enteritis), Listeria monocytogenes (L. monocytogenes), and a mixture of both before being treated with cold plasma at 70 kV for 0, 60, 120, 180, 240, and 300 s. With the extension of treatment time, the number of colonies on the surface of the fillets decreased gradually; after 300 s of cold plasma treatment, S. enteritis and L. monocytogenes populations were reduced by 2.34 log CFU/g and 1.69 log CFU/g, respectively, and the a* value and immobile water content decreased significantly (p < 0.05), while the free water content increased significantly (p < 0.05). TBARS value increased significantly (p < 0.05) to 1.83 mg MDA/kg for 300 s treatment. The carbonyl value and sulfhydryl value of sarcoplasmic protein significantly (p < 0.05) increased and decreased, respectively, as treatment time extension, while no significant changes were found in myofibrillar protein. No significant differences were observed in pH, b* value, elasticity, chewiness, thiol value, and TVB-N value. The results showed that cold plasma had an inactivation effect on tilapia fillets and could preserve their original safety indicators. It was concluded that CP treatment could be used as an effective non-thermal method to maintain the quality of tilapia fillets and extend their shelf-life.