The rise in multidrug-resistant (MDR) ESKAPE bacteria have become a major therapeutic challenge globally. Recently, novel cold atmospheric pressure plasma (CAP) as an antimicrobial is becoming popular. In this study, an indigenously developed AC cold atmospheric pressure plasma jet (CAPJ) fed with argon gas was used to evaluate its antimicrobial efficacy on these bacteria isolated from clinical specimens such as urine, blood, and sputum in a tertiary care hospital in India. The difference in CAP’s antimicrobial activity on Gram-negative bacilli (MDR E. coli) and Grampositive cocci (MDR S. aureus) was observed with various input parameters, such as microbial concentration, CAP exposure time, and exposure distance. It was observed that oxidative stress induced by reactive oxygen and nitrogen species (O₂^-, NO⁺, OH^., H₂O₂, ONOO^-, NO^., NO₂^., HO₂^., O₃^-, etc.) and electrostatic stress by ions (Ar⁺, O⁺, O₂^-, OH^-, NO⁺, OH⁺, NO₃^-, NO₂^-, O₃^-, etc.) might play a crucial role in microbial inactivation. In addition to this, a decrease in adenosine triphosphate concentration post-CAP exposure in a liquid media suggested an efficient microbial inactivation effect. The outcome of this research would be extremely beneficial to multidisciplinary researchers in this field.