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The inactivation mechanism on multidrug-resistant Acinetobacter baumannii biofilms by atmospheric-pressure helium pulse discharge plasma jet and the effect on the biofilm regeneration capacity were studied. The initial total bacterial population was 7.37 ± 0.16 log10 colony forming unit (CFU)/mL in biofilms and about 99% of the A. baumannii biofilm cells lost their cultivability after 30 min of plasma treatment. Meanwhile, resazurin fluorescent staining method showed that about 80% of the biofilm cells lost their metabolic capacity, and some bacteria entered a viable but nonculturable state. In addition, 38.3% of the bacteria lost their membrane integrity in the light of SYTO 9/propidium iodide (PI) staining assay. According to H2DCFDA fluorescent staining, the concentration of reactive oxygen species (ROS) in bacteria after plasma exposure tended to initial state after a short increase. Moreover, in the study of the regeneration ability of A. baumannii after plasma exposure for 30 min, the total bacterial number after regeneration was 5.36 ± 0.20 log10 CFU/mL. Compared with the total number of cultivable bacteria in the first-generation biofilm, about 99.99% of the bacteria were inhibited. In comparison, the biomass of the regenerated biofilm decreased to (11.0 ± 1.0)%. Meanwhile, the percentage of metabolic bacteria decreased to (52.9 ± 1.0)%. These results indicated that atmospheric-pressure helium plasma jet could not only effectively inactivate bacteria in biofilms, but also inhibit the formation of the next generation biofilms. [ABSTRACT FROM AUTHOR]