Escherichia coli, Bacillus atrophaeus, and Bacillus atrophaeus spores were exposed to a downstream plasma afterglow plume emitted from a slotted plasma device operating in open air at atmospheric pressure. The reactor electrodes were RF powered at 13.56 MHz to excite a mixture of argon and oxygen gases by a capacitive discharge as it flowed past the electrodes into open air. Bacterial inactivation experiments on surfaces exposed to the plasma afterglow were conducted with varying plasma exposure times. Experimental results demonstrated a colony forming unit (CFU) reduction of almost 5 log10 of E. coli with only 1 s of exposure per unit area. One log CFU reduction was observed in B. atrophaeus with the same treatment time of 1 s per unit area. B. atrophaeus spores showed a reduction of 3 log10 with exposure time of 10 min. Comparison on various growth media suggests that cells are killed rather than sublethally injured, while the mechanistic action of the plasma appears to affect both nucleic acids as well as the cell wall structure. These results present a promising means of inactivation of harmful microbes in a practical environment with an electrically grounded device that is handheld, much like a wand applicator. Results are applicable to the development of plasma sterilization tools for various environmental purposes.