## 1 Introduction  
Biofilm-associated oral diseases, which impact approximately 3.5 billion individuals globally, predominantly originate from the accumulation of oral biofilms at inaccessible stagnation sites, the removal of which poses a significant challenge [1]. Accumulated biofilms progressively transform into metabolically altered environments within the oral cavity, resulting in the formation of spatially heterogeneous microbial communities [2]. This phenomenon disrupts the dynamic stability of the oral microbiota, leading to ecological shifts that amplify the pathogenicity of oral biofilms [3]. Therefore, controlling the formation of oral biofilms is essential for preventing and managing oral diseases [4]. Although mechanical methods, such as tooth brushing or flossing, are recommended for oral biofilm removal, accessing some regions of the oral cavity using such tools is difficult owing to dental morphology. Supplementary methods, such as the use of antimicrobial mouthwashes, have also been used to control oral biofilm accumulation. However, prolonged or excessive use has been connected to various side effects, such as oral irritation and hyposlavitation with alcohol-based mouthwashes, and tooth discoloration, altered taste sensation, and oral mucosal irritation with chlorhexidine gluconate (CHX)-based mouthwashes [5,6]. Moreover, the indiscriminate use of antimicrobial mouthwashes may disrupt the homeostatic balance between oral microorganisms and lead to the proliferation of resistant bacteria or fungi [7,8]. Therefore, novel technologies that could selectively target clinical sites and simultaneously inhibit biofilm accumulation must be developed to reduce the incidence of adverse effects associated with antimicrobial treatment. Cold atmospheric plasma (CAP), a partially ionized gas comprising a mixture of neutral atoms, molecules, ions, and free electrons produced under conditions of atmospheric pressure and room temperature, is distinct from other plasmas that typically require high temperatures [9]. This technology has attracted interest in the dental field in recent years owing to its demonstrated safety in living tissues [10]. CAP jet, a technique used for CAP generation, can produce plasma at a certain distance from electrodes, facilitating localized application to the target area. CAP has been used for various purposes in dentistry, such as sterilization and disinfection, biofilm removal, the surface modification of dental implants, tooth whitening, oral lesion treatment, and root canal disinfection [11]. CAP produces reactive species, such as reactive oxygen and nitrogen species (RONS), that can disrupt and inactivate microorganisms, thereby facilitating biofilm removal [10; 12]. A previous study reported the presence of cytoplasmic efflux in _Streptococcus mutans_ following exposure to nitrogen-based CAP for 2 or 10 min [13]. This phenomenon was attributed to the RONS generated during the CAP application inducing cell wall erosion or cell membrane disruption through substantial electrostatic forces [8]. Furthermore, RONS can stimulate the oxidation of integral bacterial components, such as polysaccharides, proteins, lipids, and nucleic acids [12]. The antibacterial efficacy of CAP has been established in previous studies using single- and multispecies biofilm models [12; 14; 15]. One report found that argon-based CAP brush application to _Streptococcus_ or _Lactobacillus_ biofilms for 60 s resulted in complete sterilization [16]. Similarly, another study reported a significant reduction in colony-forming units (CFUs) that was observed in multispecies biofilms following argon-based CAP treatment for up to 600 s [17]. However, the clinical relevance of such biofilm models is limited due to the complexity of the oral microbial ecosystem, which comprises over 1000 different bacteria species [18]. Thus, the accurate evaluation of the antibacterial effects of CAP in biofilm models that mimic the compositional conditions of biofilms in the oral cavity, rather than specific species, is necessary.