Although various oral pathogens are inactivated by non-thermal atmospheric pressure plasma (NTAPP), the in vivo effects of NTAPP are poorly understood. The first aim of this study was to examine the antibacterial activity of microwave-pulsed NTAPP against Staphylococcus aureus in artificial saliva to mimic oral environmental conditions. The second aim was to determine the influence of microwave-pulsed NTAPP on human gingival fibroblasts (HGFs). The microwave-pulsed NTAPP reduced bacterial viability (as measured by colony forming units [CFU]) to a greater extent in artificial saliva than in saline. Extending the post-treatment incubation time increased bacterial inactivation in artificial saliva compared to saline. HGFs viability was unaffected by microwave-pulsed NTAPP for bacterial inactivation. Rather, HGFs proliferation increased after a 5-min microwave-pulsed NTAPP. Less tumor necrosis factor alpha was released by microwave-pulsed NTAPP-treated HGFs stimulated with lipopolysaccharide (LPS) than by untreated, LPS-stimulated HGFs; thus, plasma appeared to suppress the inflammatory response. Our study suggests that microwave-pulsed NTAPP may have stronger in vivo antibacterial activity than in vitro activity, and that microwave-pulsed NTAPP may have the additional advantage of suppressing gingival inflammatory responses.