Abstract The aim of this work was to investigate the efficacy of dielectric barrier discharge atmospheric cold plasma (DBD ACP) against bacteria associated with grains quality and safety. ACP inactivation efficacy was tested against biofilms formed by different strains of E. coli , Bacillus and Lactobacillus in grain model media and against B. atrophaeus endospores either in grain media or attached on abiotic surfaces. Effects were dependent on bacterial strain, media composition and mode of ACP exposure. ACP treatment for 5 min reduced E. coli spp., B. subtilis and Lactobacillus spp. biofilms by > 3 log 10 , whereas insignificant reductions were achieved for B. atrophaeus . ACP treatment of 5–20 min reduced B. atrophaeus spores in liquids by > 5 log 10 . Treatment for 30 min reduced spores on hydrophobic surface by > 6 log 10 , whereas maximum of 4.4 log reductions were achieved with spores attached to hydrophilic surface. Microscopy demonstrated that ACP caused significant damage to spores. In package ACP treatment has potential to inactivate grain contaminants in the form of biofilms, as well as spores and vegetative cells. Industrial relevance This study demonstrates that ACP technology is a promising tool for effective bio-decontamination which offers a wide range of possible applications including inactivation of microorganisms on cereal grains. However, due to the nature of the microbial contamination of grains and complex grain structures it may be necessary to optimise the potential for surface inactivation at several stages of grain processing and storage to enhance ACP efficacy against bacterial endospores.