Background Caffeic acid phenethyl ester (CAPE), a natural phenolic compound, has demonstrated antibacterial effects. Dental caries etiology is multifactorial, including a cariogenic biofilm containing multispecies bacteria. However, the antibacterial property of CAPE on multispecies biofilm is unclear. The aim of this study was to assess the effect of CAPE on the formation and cariogenicity in biofilm containing Streptococcus mutans, Streptococcus oralis, and Streptococcus mitis. Methods S. mutans (ATCC 25175), S. oralis (ATCC 35037), and S. mitis (ATCC 49456T) were employed in this investigation. Each bacterial strain was cultured in the presence of CAPE, followed by susceptibility assessment through optical density measurements at a 600 nm wavelength. Multispecies biofilm formation was achieved by co-culturing S. mutans, S. oralis, and S. mitis at a 1:1:1 ratio on hydroxyapatite-coated 96-well plates. The anti-adherence activity of CAPE on multispecies biofilm was evaluated using a crystal violet staining assay. Cariogenic gene expression level and glucosyltransferase (GTF) function in CAPE-treated mixed bacteria were evaluated using real-time PCR and enzyme activity assay, respectively. The thickness and bacterial viability in CAPE-treated multispecies biofilm were examined using confocal laser scanning microscopy. Results CAPE demonstrated a significant antimicrobial effect on S. mutans, S. oralis, and S. mitis (p < 0.05). The inhibition concentration 50% (IC50) of CAPE against S. mutans, S. oralis, and S. mitis ranged from 1.6–6.4 mg/ml. CAPE significantly hindered the multispecies biofilm adherence (p < 0.05). Furthermore, the expression of genes involved in acidogenicity, aciduricity, sucrose-dependent adhesion and quorum sensing mechanism and GTF activity were significantly decreased in CAPE-treated mixed bacteria (p < 0.05). In a multispecies biofilm, CAPE significantly reduced its thickness and viable bacteria population (p < 0.05). In conclusion, CAPE exhibited antimicrobial, anti-adherence and anti-cariogenic effects within a multispecies biofilm. These findings suggest the potential use of CAPE as an adjunctive anti-cariogenic agent in future dental applications.