B Background: dental caries is one of the most prevalent oral health problems in indonesia and is closely associated with microorganisms such as staphylococcus aureus and candida albicans. hydroxyapatite (ha) derived from bovine cancellous bone contains high mineral content, exhibits good biocompatibility, and has the potential to be used as a natural active ingredient in toothpaste to inhibit microbial growth without increasing enamel surface roughness. objective: to analyze the effect of hydroxyapatite-based toothpaste at concentrations of 1%, 3%, and 5% on the inhibition of s. aureus and c. albicans, as well as changes in enamel surface roughness. methods: after formulating ha toothpaste at concentrations of 1%, 3%, and 5%, antimicrobial testing was performed using the well diffusion method, with chloramphenicol 100 ppm used as an antibacterial reference control against s. aureus and nystatin 5,000 mg/l used as an antifungal reference control against c. albicans. enamel surface roughness was measured using a surface roughness tester at baseline, and after 28, 56, and 84 minutes of brushing. results: the 5% ha toothpaste produced the greatest inhibitory effect against staphylococcus aureus (9.1 ± 0.1 mm), whereas the 1% ha toothpaste showed the greatest inhibitory effect against candida albicans (9.7 ± 1.2 mm). surface roughness increased with increasing ha concentration. however, at all ha toothpaste concentrations of 1%, 3%, and 5%, the roughness values remained within the safe limit (<0.2 µm). conclusion: the results indicate that the 5% ha toothpaste is the most effective formulation, as it provides strong antimicrobial activity and remains safe for use without causing an increase in enamel roughness exceeding the clinical threshold of 0.2 µm.