Healthcare-associated infections (HCAI) affect 5–10% of acute hospital admissions. Environmental decontamination is an important component of all strategies to prevent HCAI as many bacterial causes survive and persist in the environment, which serve as ongoing reservoirs of infection. Current approaches such as cleaning with detergents and the use of chemical disinfectant are suboptimal. We assessed the efficacy of helium and helium–air plasma in killing Staphylococcus aureus and Clostridium difficile on a glass surface and studied the impact on bacterial cells using atomic force microscopy (AFM). Both plasma types exhibited bactericidal effects on Staph. aureus (log3·6 – >log7), with increased activity against methicillin-resistant strains, but had a negligible effect on Cl. difficile spores (<1log). AFM demonstrated cell surface disruption. The addition of air increased the microbicidal activity of the plasma and decreased the exposure time required for an equivalent log reduction. Further evaluation of cold plasma systems is warranted with, for example, different bacteria and on surfaces more reminiscent of the health care environment as this approach has potential as an effective decontaminant.