Candida albicans colonizes sites with different environmental pH. However, it is unclear how these conditions can interfere on biofilms. This study aimed to evaluate the influence of environmental pH on behavior of C. albicans regarding its structure, virulence and susceptibility to fluconazole (FLZ). Minimal inhibitory concentration, minimal fungicidal concentration and time kill were used to evaluate the susceptibility to FLZ in planktonic cells under three pH values (4.0, 5.5, 7.0). These pH values were used for biofilms analysis. C. albicans ATCC 90028 was developed on poly(methlymethacrylate) resin for 48 h. Then, 2.56 μg/mL of FLZ was added to experimental groups for 24 h, and biofilms were analyzed by cell quantification, bioactivity, secretion of proteinases and phospholipases and structure. All data were analyzed by two-way ANOVA, followed by Tukey's test (α = 0.05). For planktonic cells, changes in environmental pH decreased the susceptibility to FLZ. C. albicans biofilms developed at pH 5.5 showed higher cell counts, bioactivity, bio-volume, average thickness and roughness coefficient (p < 0.05). In contrast, the presence of FLZ at pH 4.0 did not influence the structural parameters (p > 0.05), but increased secretion of proteinase and phospholipase (p < 0.05). Within the conditions studied, it was shown that environmental pH modulates the structure, virulence and susceptibility of C. albicans to FLZ.69-7039-44