The present study demonstrates the antifungal potential of chemically characterized essential oil (EO) of Blume on spp. biofilm and establishes its mode of action, effect on fungal growth kinetics, and cytotoxicity to human cells. The minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) values varied from 62.5 to 1,000 g/mL, and the effect seems to be due to interference with cell wall biosynthesis. The kinetics assay showed that EO at MICx2 (500 g/mL) induced a significant (p < 0.05) reduction of the fungal growth after exposure for 8 h. At this concentration, the EO was also able to hinder biofilm formation and reduce spp. monospecies and multispecies in mature biofilm at 24 h and 48 h (p < 0.05). A protective effect on human red blood cells was detected with the EO at concentrations up to 750 g/mL, as well as an absence of a significant reduction (p > 0.05) in the viability of human red blood cells at concentrations up to 1,000 g/mL. Phytochemical analysis identified eugenol as the main component (68.96%) of the EO. Blume EO shows antifungal activity, action on the yeast cell wall, and a deleterious effect on spp. biofilms. This natural product did not show evidence of cytotoxicity toward human cells.