Aims and Objectives: To evaluate and compare the antifungal efficacy of low molecular weight chitosan and nystatin by their inhibitory action on candida biofilm formation on Triplex heat cure denture base after 7 days and 14 days. Materials and Methods: A total of 10 specimens were fabricated using the Triplex heat cure PMMA acrylic resin by the conventional compression molding technique.Candida albicans biofilm was allowed to form over a period of 72 hours on all the specimens after pre-treatment with artificial saliva. Two groups of 5 specimens each were established. Group A was treated with low molecular weight chitosan suspension and group B was treated with nystatin suspension, both on the 4th day after biofilm formation. The treated specimens were now stored in a yeast nitrogen base broth medium to complete the time period of 7 days for initial maturation of the biofilm. The CFU count of candida colonies was recorded on the 7th day in both the groups.Specimens were treated again on the 11th day using chitosan and nystatin suspension for the assigned groups and the residual biofilm was allowed to completely mature till the 14th day, when another CFU count of the candida colonies was recorded for respective groups. The results obtained were compared and subjected to statistical analysis. Results: The results showed that there was a significant reduction in the number of candida colonies in group A (Chitosan) from the 7th to 14th day of biofilm formation. (p value = 0.04). On the other hand, the mean count of candida colonies showed insignificant reduction from 7 th to 14th day in group B (nystatin) with a p value of 0.20. On comparison of CFU count between Group A and B on 7th day, insignificant reduction was witnessed (p value = 0.26) but a markedly significant decrease in the number of colonies was seen on the 14th day (p value = 0.02). Conclusion: It was concluded thatChitosan possesses noticeably better antifungal properties as compared to nystatin, especially when it comes to inhibitory effect on mature and adherent candida albicans biofilm formation on heat cured PMMA acrylic resin denture bases after 14 days.