Acinetobacter baumannii is an intrinsically multidrug-resistant pathogen that, when existing as a biofilm, confers increased environmental tolerance to desiccation, nutrient starvation as well as increased tolerance to antimicrobials. Outbreaks of A. baumannii infections within the clinical setting are often associated with the biofilm phenotype. This study investigates the role of biofilm biomass in A. baumannii susceptibility to exposure to a kilohertz-driven, in-house-designed, cold plasma jet, through the examination of cold plasma treatment efficacy in A. baumannii biofilms grown over various times for up to 72 h. For biofilms grown for 24, 48 and 72 h, D values were 19·32 ± 2·71, 29·18 ± 3·15 and 24·70 ± 3·07 s respectively. Monitoring A. baumannii biofilm biomass over these time periods revealed that the greatest biomass was observed at 48 h with the lowest biofilm biomass at 24 h growth. Enumeration of viable biofilm colony counts at each time point was comparable. Scanning electron microscopy images of plasma-treated biofilms revealed extensive surface damage of A. baumannii cells. These results describe the role of biomass in mediating A. baumannii biofilm susceptibility to cold plasma treatment, implicating the biofilm matrix as a protective barrier to the antimicrobial effects of cold plasma. SIGNIFICANCE AND IMPACT OF THE STUDY: Acinetobacter baumannii biofilm formation results in increased environmental and antimicrobial tolerance and resistance compared to the planktonic phenotype. Cold plasma technology is increasingly investigated as a new tool for decontamination of biofilm-contaminated surfaces, especially those found in the clinical setting. This new technology presents a promising approach to the remediation of surfaces contaminated by biofilms. This study identifies the role played by A. baumannii biofilm biomass in mediating tolerance and susceptibility to cold plasma treatment. This work demonstrates that increased biofilm biomass reduces the efficacy of antimicrobial species generated by cold plasma, resulting in greater tolerance to plasma exposure.