Background and aim: CHITOSAN has the property of inhibition of the growth of many pathogenic bacteria and fungi. Tissue conditioners are good substances for the growth and colonization of a variety of micro-organisms. The aim of this research was to investigate the antibacterial and antifungal properties of CHITOSAN, ZnO and Ag nanoparticles added to tissue conditioners. Material and Method: In this clinical trial study, 42 patients were randomly divided into 7 groups. In first group, the patients received the tissue conditioners (control group) and the second to seventh groups received the tissue conditioners included CHITOSAN, ZnO and Ag by weight percentage of 0. 675, 1. 25, 2. 5, 5, 10, 20. After 24 and 48 hours using these substances, the microbial culture inside the mouth and denture was done by sterile swap. Results: Complete inhibition of growth at 24 h and 48 h occurred in Candida albicans at 2. 5 % concentration and in Enterococcus faecalis, Pseudomonas aeruginosa and Streptococcus mutans at 5% concentration of CHITOSAN, ZnO and Ag nanoparticles. Over time, the growth rate of Candida albicans, Enterococcus faecalis, Pseudomonas aeruginosa and Streptococcus mutans was decreased. Conclusion: Increasing the concentration of nanoparticles increases the growth inhibition in all studied microorganisms. By increase of growth inhibition, the growth of microorganisms also increases. An enhancement of 5% in the concentration of tissue conditioners results in complete inhibition of the studied microorganisms.