Background and Aim : The activity of bacteria that cause tooth decay is increasing. The use of antimicrobial properties of natural materials based on effective dental compounds is one of the future hopes in the microbiology of oral diseases. Oral & dental pathogenic bacteria such as Lactobacillus and Streptococcus mutans strains can cause dental lesions in both planktonic and biofilm phases. The aim of this study was to investigate a chemically compound of Chitosan-zinc oxide (N-Ch-ZnO) against the Lactobacillus acidophilus (LA) and Streptococcus mutans (SA) which are synthesized in a nanoparticle size.Methods : The Broth Dilution Test (BDT) was used to evaluate the antibacterial properties in Planktonic and the Microtiter Plate Method (MPM) in Biofilm phase .The studied strains were SA (ATCC ۳۵۶۶۸) and LA (biofilm-producing strain). A chemical compound based on MIC value of Chitosan and ZnO for each tested bacteria (Planktonic) and MBEC value (in Biofilm phase) were used in proportion of ۵۰%. The LA were cultured on MRS medium and SA on BHI medium for ۴۸ hours incubation with ۵% Co۲. Chlorhexidine was used as a positive and sterile distilled water was used as a negative control. A total of ۳۰ times for Planktonic for each bacteria and ۳۰ times the biofilm phase was tested. The experimental results were analyzed by ANOVA.Results : The results showed that the diameter of the LA growth inhibition zone was ۱۴ mm compared to the ۲۰ mm zone of Chlorhexidine. There was no inhibition zone for SA. In the biofilm phase, the compound of N-Ch-ZnO could not eradicated the biofilm form each of the bacteria.Conclusion : This study showed that the compound of N-Ch-ZnO was significant relationship in inhibiting LA planktonic phase but not in biofilm phase. There was no significant relationship between SA in either planktonic or biofilm forms. This research indicated that the ۵۰% proportion of Chitosan and ZnO was not proper for biofilm eradication. Therefore is need to more study in other ratios in both form in theses to bacteria is recommended.