Cold atmospheric pressure plasma jet (CAPJ) has piqued the interest of researchers for various antimicrobial applications such as disinfection, wound decontamination, etc. In the current context, a deeper understanding of the correlation between CAPJ's intrinsic parameters, discharge characteristics, species composition, and antimicrobial activity is required for any successful application. This research evaluated the effect of intrinsic operational parameters such as voltage, frequency, gas flow rate, and operating gas on the reactive species composition of an in-house-developed CAPJ discharge along with the antimicrobial activity. It was observed that the identified excited atoms (Ar I, He I, N2, and O I), ions (Ar+, N2+, N+, H2O+, H3O+, etc.), radical reactive oxygen and nitrogen species (RONS) (OH•), and nonradical RONS (O I, O+, OH+, NO+, O2+, O2−, NO2−, N2O2−, NO3−, N2O3−, etc.) might play a synergistic role in bacterial inactivation via oxidative and electrostatic stress. The variation in voltage, frequency, gas flow rate, and operating gas influenced the discharge chemistry, leading to variation in bacterial inactivation. The reactive species in the discharge responsible for such variation was evaluated extensively. This investigation into various operational parameters would aid in determining the most effective settings for a developed CAPJ to achieve high productivity.