Background: Denture stomatitis is highly prevalent in denture wearing patient, & microbial plaque accumulation on intaglio surface of removable dentures plays a critical role in formation of denture biofilm. The purpose of this study was to evaluate the growth of candidal species on Denture Biofilm using new sampling technique in vivo & testing efficacy of Chlorhexidine as disinfectant on denture biofilm of patients with & without denture stomatitis. Methods: Patients wearing complete dentures with partial denture stomatitis as study group & patients without denture stomatitis as control group were taken. Separate acrylic resin disks were inserted at denture stomatitis lesion site and at normal mucosal sites coinciding with the dentures of study group & control group. After 24 hours and 3 days of wear of dentures, the disks were retrieved and compared for number of candidal colony forming unit (CFU)s per ml of samples & efficacy of 0.5% Chlorhexidine (CHX-0.5%) on same samples from the lesion & normal mucosal sites respectively. Results: The No. of Candidal (CFU)s count on denture biofilm increased exponentially from day-1 to day-3 with statistically highly significant order at lesion site & with statistically significant order at non-lesion site of denture stomatitis patients. 0.5% Chlorhexidine was found to be very effective & of statistically highly significant order in reducing the fungal load at lesion site of denture stomatitis. Conclusion: The complete dentures can be soaked in CHX-0.5% for specific time period to treat or to avoid denture stomatitis in denture wearing patients.