Polymethylmethacrylate (PMMA) is widely used in the fabrication of dentures due to its aesthetic appeal and mechanical strength. However, PMMA's susceptibility to microbial colonization often leads to oral infections such as denture stomatitis. Enhancing the antimicrobial properties of denture materials is crucial for improving patient outcomes.Chitosan, a natural biopolymer, possesses inherent antimicrobial properties and could potentially enhance the microbial resistance of PMMA. This study has investigated thepotential of chitosan-reinforced heat-polymerized PMMA denture material to reduce microbial colonization. The aim of the study was to evaluate and assess the anti-bacterial and antifungal properties of chitosan-reinforced heat-polymerized PMMA with conventional heat-polymerized PMMA Materials and methods:Chitosan-reinforced PMMA samples were fabricated with varying chitosan concentrations (0% control, 5%, 10%, and 15% by weight). The fabrication involved mixing chitosan powder with PMMA powder, adding monomer liquid, followed by mixing, packing, and curing using the conventional heat polymerization technique. The antimicrobial efficacy was assessed in vitro using two common oral pathogens: Streptococcus mutans and Candida albicans. Blood agar plates were used for S. mutans and Sabouraud agar plates were used for C. albicans. Each sample was placed on the respective agar plates inoculated with a standardized microbial suspension and incubated at 37°C for 24 hours. The number of colony-forming units (CFUs) was counted to quantify microbial growth. Statistical analyses, including linear regression analysis, one-way ANOVA test, and Pearson correlation were performed to evaluate the relationship between chitosan concentration and antimicrobial efficacy. The p-value was calculated to determine the statistical significance of the results. The chitosan-reinforced PMMA samples showed significantly greater antimicrobial efficacy compared to the conventional PMMA samples. The CFU counts for both S. mutans and C. albicans decreased with increasing chitosan concentration. Linear regression analysis indicated a strong negative correlation between chitosan concentration and CFU counts, with Pearson correlation coefficients of -0.97 for S. mutans and -0.98 for C. albicans. ANOVA analysis revealed a statistically significant difference in antimicrobial efficacy across different chitosan concentrations (p < 0.001). Incorporating chitosan into heat-polymerized PMMA significantly enhances its antimicrobial properties against S. mutans and C. albicans. The antimicrobial efficacy improves with higher concentrations of chitosan, with the 15% chitosan-reinforced samples showing the most substantial reduction in microbial growth. These results suggest that chitosan-reinforced PMMA dentures could be a superior alternative to conventional PMMA dentures, potentially reducing denture-related infections and improving oral health outcomes for denture wearers.