Recently, it was suggested that the nitrite (NO2−) produced from NO3− by oral bacteria might contribute to oral and general health. Therefore, we aimed to clarify the detailed information about the bacterial NO2-production in the oral biofilm. Dental plaque and tongue-coating samples were collected, then the NO2-producing activity was measured. Furthermore, the composition of the NO2−-producing bacterial population were identified using the Griess reagent-containing agar overlay method and molecular biological method. NO2−-producing activity per mg wet weight varied among individuals but was higher in dental plaque. Additionally, anaerobic bacteria exhibited higher numbers of NO2−-producing bacteria, except in the adults’ dental plaque. The proportion of NO2−-producing bacteria also varied among individuals, but a positive correlation was found between NO2−-producing activity and the number of NO2−-producing bacteria, especially in dental plaque. Overall, the major NO2−-producing bacteria were identified as Actinomyces, Schaalia, Veillonella and Neisseria. Furthermore, Rothia was specifically detected in the tongue coatings of children. These results suggest that dental plaque has higher NO2−-producing activity and that this activity depends not on the presence of specific bacteria or the bacterial compositions, but on the number of NO2−-producing bacteria, although interindividual differences were detected.