| Summary: | The characteristics of the denture base surface, in combination with the oral environment, promote the colonization and development of <i>Candida albicans</i> biofilm, which is the main cause of denture stomatitis. This study evaluated the effectiveness of fibrin biopolymer with digluconate chlorhexidine or <i>Punica granatum</i> alcoholic extract to prevent <i>C. albicans</i> biofilm. Conventional heat polymerized and pre-polymerized poly(methyl methacrylate) (PMMA) circular specimens (10 × 2 mm) were fabricated (<i>n</i> = 504) and randomly divided into groups: no treatment (control—CT), fibrin biopolymer coating (FB), fibrin biopolymer with <i>P. granatum</i> (FBPg), or digluconate of chlorhexidine (FBCh) coating. The specimens were inoculated with <i>C. albicans</i> SC5314 (1 × 10<sup>7</sup> cells/mL) and incubated for 24, 48, and 72 h. Crystal violet and colony-forming unit assays were used to quantify the total biofilm biomass and biofilm-living cells. A qualitative analysis was performed using confocal laser scanning microscopy. Data obtained are expressed as means and standard deviations and were statistically analyzed using a three-way analysis of variance (α = 0.05). The FBPg and FBCh groups inhibited the growth of <i>C. albicans</i> biofilm in both PMMA materials analyzed, with FBCh performing better in all periods evaluated (<i>p</i> < 0.0001). The colony forming unit (CFU) assay showed that the FB group favored the <i>C. albicans</i> biofilm growth at 24 h and 48 h (<i>p</i> < 0.0001), with no differences with CT group at 72 h (<i>p</i> = 0.790). All groups showed an enhancement in biofilm development up to 72 h (<i>p</i> < 0.0001), except the FBCh group (<i>p</i> = 0.100). No statistical differences were found between the PMMA base materials (<i>p</i> > 0.050), except in the FB group (<i>p</i> < 0.0001). Fibrin biopolymer, albeit a scaffold for the growth of <i>C. albicans,</i> when combined with chlorhexidine digluconate or <i>P. granatum</i>, demonstrated excellent performance as a drug delivery system, preventing and controlling the formation of denture biofilm.
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