ABSTRACT: This study evaluated the susceptibility of Streptococcus mutans (ATCC 25175) and 9 clinical isolates from Thai children to different formulations of Citrus hystrix essential oils. All strains were incubated for 3 and 6 h to form biofilms suitable for susceptibility tests. This study also evaluated the inhibitory effect of each oil formulation on growth and biofilm formation of S. mutans (ATCC 25175) using a 96-well polystyrene plate biofilm assay, and analysed the selected formulations that showed the most inhibitory effect from the susceptibility and biofilm assay tests and 3 clinical isolations for further evaluation using fluorescent dyes (SYTO9 and propidium iodide) and confocal laser scanning microscopy (CLSM). Results show that all strains of S. mutans tested in this study were sensitive to most of the oil formulations (≥99.9%) when compared to the controls. Most of the oil formulations had an inhibitory effect on 3 h and 6 h biofilm formation (≥50%) compared to the control. Most of the oil formulations inhibited biofilm formation of the standard strain and all clinical isolates in biofilm assay. All formulae of the essence from the leaf and fruit of C. hystrix oil exhibited a significant (p<0.05) inhibitory effect on S. mutans and all clinical isolations when compared to the control group and visualized by CLSM. Thus C. hystrix essential oils might be an alternative, inexpensive natural medication to prevent dental caries.