The level of dental and oral hygiene on elementary school age children is very low, so it can be increasing the incidence of dental and oral diseases which caused by various microorganisms. Mouthwash can keeping dental and oral hygiene, but many commercial mouthwash is still used alcohol as their ingredients, which can be giving side effects, therefore, we still needed used alternative mouthwash, which made from nano chitosan and nano calcium, derived from nature Xylotrupes Gideon. Methods: This is a experimental study with pre and post test control group design using total plate count method. Total is 27 subjects, aged 9 years. Processing data is done by statistical test Chi Square, and one way ANOVA with software of SPSS 23. Results: Results showed that serum concentration of glucose, cholesterol and triglyceride were significantly higher (p ≤ 0.05) when compared with the fluid from three follicles size categories (Small, Medium and large follicles in CL + and CLovaries). The differences between follicle size categories in CLovaries were only significant for concentrations of glucose and triglyceride. The FF concentration of glucose and cholesterol in same follicle size categories (Small, Medium and large follicles in CL + and CLovaries) were significant (P ≤ 0.05). Conclusions: Measurement results the quantity of bacteria suggested that decreased of bacteria colonies before and after gargle, on negative control 30%-73%, positive control 33%-100%, the 250ppm concentration is 95% -100%, 500ppm and 750 ppm concentration is 100%. Nanoparticles of chitosan and calcium-based mouthwash formulation from X. gideon has the ability to reduce oral bacterial colonies quantity among elementary school age children. More high concentrations of mouthwash formulations which are used more low quantity of oral bacteria colonies.