The worrying rise in antibiotic resistances emphasizes the need to seek new approaches for treating and preventing periodontal diseases. The purpose of this study was to evaluate the antibacterial and anti-biofilm activity of cranberry in a validated in vitro biofilm model. After chemical characterization of a selected phenolic-rich cranberry extract, its values for minimum inhibitory concentration and minimum bactericidal concentration were calculated for the six bacteria forming the biofilm (Streptococcus oralis, Actinomyces naeslundii, Veillonella parvula, Fusobacterium nucleatum, Porphyromonas gingivalis, and Aggregatibacter actinomycetemcomitans). Antibacterial activity of the cranberry extract in the formed biofilm was evaluated by assessing the reduction in bacteria viability, using quantitative polymerase chain reaction (qPCR) combined with propidium monoazide (PMA), and by confocal laser scanning microscopy (CLSM), and anti-biofilm activity by studying the inhibition of the incorporation of different bacteria species in biofilms formed in the presence of the cranberry extract, using qPCR and CLSM. In planktonic state, bacteria viability was significantly reduced by cranberry (p < 0.05). When growing in biofilms, a significant effect was observed against initial and early colonizers (S. oralis (p 0.017), A. naeslundii (p = 0.006) and V. parvula (p = 0.010)) after 30 or 60 s of exposure, while no significant effects were detected against periodontal pathogens (F. nucleatum, P. gingivalis or A. actinomycetemcomitans (p > 0.05)). Conversely, cranberry significantly (p < 0.001 in all cases) interfered with the incorporation of five of the six bacteria species during the development of 6 h-biofilms, including P. gingivalis, A. actinomycetemcomitans, and F. nucleatum. It was concluded that cranberry had a moderate antibacterial effect against periodontal pathogens in biofilms, but relevant anti-biofilm properties, by affecting bacteria adhesion in the first 6 h of development of biofilms