| Summary: | (1) Background: <i>Enterococcus faecalis</i> and <i>Candida albicans</i> are often isolated from infected root canals. The interaction between these two species is not clear enough. Therefore, the objective of this study was to investigate the effect of <i>E. faecalis</i> on the biofilm formation, hyphal morphogenesis and virulence gene expression of <i>C. albicans</i>. (2) Methods: We used the culture supernatant of <i>E. faecalis</i> (CSE) to treat the biofilms of <i>C. albicans</i>. Then, crystal violet staining and colony-forming unit (CFU) counting were performed to evaluate biofilm biomass. Scanning electron microscopy (SEM) and confocal laser scanning microscope (CLSM) were applied to observe fungal morphology. Subsequently, exopolymeric substances (EPS) production, cellular surface hydrophobicity (CSH) and adhesion force of biofilms were investigated by CLSM, water–hydrocarbon two-phase assay and atomic force microscopy (AFM), respectively. Finally, the expression of <i>C. albicans</i> virulence genes (<i>ALS1</i>, <i>ALS3</i>, <i>HWP1</i> and <i>EFG1</i>) were measured by RT-qPCR assay. (3) Results: The exposure of CSE promoted the biofilm formation and hyphal morphogenesis of <i>C. albicans</i>, increased the EPS production, CSH and adhesion force of <i>C. albicans</i> biofilms, and increased the expression level of <i>EFG1</i>. (4) Conclusions: Our data indicated that CSE promoted the hyphal morphogenesis and biofilm formation of <i>C. albicans.</i>
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