The aim of this study is to examine the effect of nano hydroxyapatite in the toothpaste and its effect on the pH and microbial activity. Nano-hydroxyapatite (nHAp) is considered one of the materials that have high biocompatibility for biomimetic material due to its chemical and morphological similarity with dental apatite. Additionally, it has been documented to possess antibacterial potentials. The present study was conducted to identify the relationship between oral microenvironment pH change and its role in the Streptococcus mutans viability, a common pathogen in the oral cavity. Change in pH is closely related to number of Streptococcus mutans as the main cariogenic organism and acid-producing bacteria. The study was carried out using 0.25%, 0.7%, and 1.5% concentration of HAp in toothpaste formulation and commercial toothpaste as control. Our studies showed that the most significant pH fall was observed in 1.5% and 0.7% nHAp. However, in 60 minutes, all nHAp groups were able to restore pH into neutral, especially in the 0.7% nHAp, which reached a pH of 7. As a comparison, the commercial toothpaste only returned to 6 within 1 hour. Experimental method of this study is In vitro oral microenvironment pH and microbiological analysis on teeth fragment. The detailed data about microstructure and antibacterial activity will be presented.