The limited knowledge of plasma-activated media (PAM) antimicrobial effects hampers applications of cold atmospheric plasmas for wastewater treatment. To address these limitations, the present work focuses on the study of the plasma-culture media interaction and induced bacteria inactivation. A Surface Dielectric Barrier Discharge (SDBD) is directly applied on M. luteus and E. coli resuspended in deionized water and Luria-Bertani (LB) broth. Bacteria inactivation is enhanced during 24-h post-plasma treatment time, demonstrating plasma is more energy efficient than conventional autoclave. To achieve a total bacteria inactivation of 107–108 CFU/ml in deionized water the plasma energy consumption is 0.05 kWh/l for M. luteus and 0.15 kWh/l for E. coli. In LB broth, the same total bacteria inactivation requires 0.49 kWh/l of energy compared to autoclave energy consumption of 0.58 kWh/l. The evaluation of plasma-generated reactive nitrogen species by high-pressure ion chromatography reveals the higher NO2− concentration in the treated liquid with the higher amount of organic matter. In addition, bacteria membrane integrity and changes in the membrane potential are studied by flow cytometry. The compromised membrane integrity suggests E. coli underwent cell lysis while part of the population could enter viable but non-culturable state upon plasma treatment. The presence of LB broth in the liquid prevents cell membrane depolarization due to the buffering capacity of the LB broth. This study highlights the validity of cold atmospheric plasmas application for organic wastewater decontamination.