Background: Chitosan and Calcium can be regarded as the natural polymeric materials obtained from the exoskeleton of Xylotrupes gideon through demineralization, deproteinization and deacetylation processes. Nano chitosan and calcium can inhibit the demineralization process, which is caused by the consumption of acetic acid. This study aims at determining the effect of nano chitosan pretreatment and nano calcium X. gideon on the application of acetic acid toward tooth enamel hardness. Methods: The study used 36 upper teeth premolars divided into 4 groups; the first group was artificial saliva control, the second group was acetic acid application without pretreatment with nano chitosan and nano calcium, the third group was given pretreatment with nano chitosan and the fourth group was given pretreatment with nano chitosan and nano calcium (5:1). Results: The results showed that there were significant differences in all groups both without or with nano chitosan pretreatment and combination nano chitosan and nano calcium X. gideon on the application of acetic acid to hardness of tooth enamel (p <0.05). Conclusions: Research using combination of nano chitosan and nano calcium proved to be more effective in inhibiting the demineralization process by increasing the hardness of tooth enamel as compared to other groups.