ABSTRACT In the present study, tyrosol functionalized chitosan gold nanoparticles (Chi-TY-AuNP’s) were prepared as an alternative treatment strategy to combat fungal infections. Various biophysical techniques were used to characterize the synthesized Chi-TY-AuNP’s. The antifungal and antibiofilm activities of Chi-TY-AuNP’s were evaluated against C. albicans and C. glabrata and efforts have been made to elucidate the possible mechanism of action. Chi-TY-AuNP’s showed a high fungicidal effect against both sessile and planktonic cells of Candida spp. Additionally, Chi-TY-AuNP’s completely eradicated (100%) the mature biofilms of both the Candida spp. FESEM analysis highlighted the morphological alterations in Chi-TY-AuNP’s treated Candida biofilm cells. Effect of Chi-TY-AuNP’s on the ECM components showed significant reduction in protein content in C. glabrata biofilm and substantial decrease in extracellular DNA (eDNA) content of both the Candida spp. ROS generation analysis using DCFDA-PI staining showed high ROS levels in both the Candida spp., whereas pronounced ROS production was observed in Chi-TY-AuNP’s treated C. glabrata biofilm. Biochemical analysis revealed decreased ergosterol content in Chi-TY-AuNP’s treated C. glabrata cells, while inconsequential changes were observed in C. albicans . Furthermore, the transcriptional expression of selected genes (ergosterol biosynthesis, efflux, sterol importer, and glucan biogenesis) was reduced in C. glabrata in response to Chi-TY-AuNP’s except ERG11 and CDR1 . Conclusively the result showed the biofilm inhibition and biofilm eradication efficacy of Chi-TY-AuNP’s in both the Candida spp. Findings of the present study manifest Chi-TY-AuNP’s as a potential therapeutic solution to Candida biofilm-related chronic infections and overcome biofilm antifungal resistance.