Cold atmospheric plasma (CAP) has been investigated to control local infections, such as peri-implantitis or periodontitis, since reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced from CAP have excellent antibacterial activity. Porphyromonas gingivalis (P. gingivalis) grown on titanium (Ti) disks was treated with CAP under various conditions in vitro and assessed for its antibacterial capacity. CAP apparatus was designed to treat periodontitis. P. gingivalis biofilm was formed onto the Ti disks for application in periodontitis treatment. True power of plasma jet plume from CAP apparatus gradually increased according to increase of intensity/energy level ratio. Optical emission spectroscopy (OES) of plasma jet plume from CAP equipment showed that the peaks of Ar+, OH and O ions were observed in the UV range (200 nm-400 nm) and visible range (690 nm-950 nm). In OES analysis, the peaks of OH and O ions were relatively increased when the volume of oxygen supply was decreased compared to Ar. Furthermore, the increase of plasma intensity increased the peaks of OH and O ions. P. gingivalis biofilm was formed onto the Ti disks and confirmed viability of P. gingivalis. Production of ROS in the aqueous solution by CAP apparatus gradually increased according to the treatment time and intensity of plasma. The increased ROS level induced decrease of surviving P. gingivalis according to treatment time and intensity of plasma. Furthermore, CAP treatment of P. gingivalis biofilm on the Ti disks also induced death of bacterial cells. Longer treatment time and higher energy level of CAP apparatus resulted in decrease of HGF-1 cell viability. Thus, appropriate treatment time and intensity of plasma is required to minimize cytotoxicity against normal cells with bactericidal effect. Practically, 6.387 W of plasma power and 60 s of treatment time was considered as an appropriate treatment condition to diminish normal cell cytotoxicity. We suggest that the CAP apparatus is a promising candidate to eradicate biofilm with antibacterial efficacy through ROS generation from CAP apparatus.