Purpose: The purpose of this study is to establish the acceptable intraoral application time of antiseptic agents and evaluate the effect of mechanical irrigation. Methods: A total of 80 subjects were selected for this study. Saliva secreted at the resting state was taken. The subjects were divided into 8 experimental groups, and kept 10% povidone-iodine (PVI) or 0.2% chlorhexidine gluconate (CHX) for 20 or 40 seconds in their oral cavity with/without irrigation of the oral cavity with sterilized normal saline, respectively. Then, the saliva was taken and diluted with phosphate buffered saline and then plated onto 5% sheep blood agar plates, which were incubated. Colony forming unit (CFU) was measured for the salivary bacterial counts. Results: After application of PVI and CHX, all the experimental groups showed statistically significant decrease in CFU (P<0.01). Group 2 (PVI, 40 s) showed more significant reduction rate in CFU than group 4 (CHX, 40 s; P<0.01). Group 6 (PVI, 40 s, irrigated) showed more significant reduction rate than group 2 (PVI, 40 s; P<0.01). Group 2 (PVI, 40 s) showed more significant reduction rate than group 1 (PVI, 20 s; P<0.01). Conclusion: Application of PVI for 40 seconds and mechanical irrigation with sterilized normal saline showed the best result among the 8 groups in terms of the reduction rate of salivary bacterial counts.