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Aim Methodology Results Conclusions Natural bioactive products have been tested as alternative antimicrobial agents. This study evaluated the effect of Punica granatum extract (PGE) on oral multispecies biofilms.Lyophilized extracts from pomegranate peel were prepared, and the punicalagin content was assessed by ultra‐performance liquid chromatography (UPLC). Oral multispecies biofilms from 2 donors were grown on four collagen‐coated hydroxyapatite discs. After incubation for 7 days or 3 weeks, the biofilms were exposed to water (control), 2% CHX, 10% PGE, 20% PGE or 30% PGE for 3 min. The proportions of dead bacteria were assessed by the live/dead staining and confocal microscopy. After the analysis, the best PGE concentration (30%) was combined with CHX. The experimental phases were repeated using water, 2% CHX, 30% PGE and 30% PGE + 2% CHX. Five random areas of the biofilm on each disc were scanned, resulting in 20 scanned areas for each group.Regarding the biofilm volume, no differences were found amongst solutions (p = .111). The PGE solution killed bacteria effectively in 1‐week, 2‐week and 3‐week‐old‐plaque biofilms, ranging from 37 to 55.3%, depending on the PGE concentration. The 30% PGE (a) (p = .0009) had greater antibiofilm effectiveness than 2% CHX (b), which killed bacteria in the 25.2 to 48.7% range. The 10% and 20% PGE had intermediate values (ab), without significant differences from 30% PGE (p = 1.002). Water (c) had the lowest proportion of dead bacteria (p < .00001) in a range of 5 to 6.7% and lower effectiveness in killing bacteria (p < .05). The PGE alone or mixed with 2% CHX had greater anti‐biofilm effectiveness than CHX (p < .05). The old plaque biofilms were more resistant than the 7‐day‐old plaque (p < .05).The 30% PGE (alone or combined with CHX) exhibited a greater antibiofilm effect on oral multispecies biofilms grown on hydroxyapatite discs than 2% CHX. [ABSTRACT FROM AUTHOR]