Background:The search on natural plant compounds revealed that those compounds can be used for the elimination of oral biofilms as a substitute of current chemical based treatments and is becoming of increasing interest. Objectives: The objective of this study was to investigate the biofilm inhibitory effect and disruptive efficacy of Shiitake mushroom (Lentinula edodes) extracts. Materials and Methods: Essential oils were extracted by hydrodistillation and the components were analyzed by gas chromatography mass-spectroscopy. Biofilm assessments were carried out on four oral pathogens and a mixed culture containing Streptococcus mutans, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. Minimum inhibitory concentrations (MIC), minimum bactericidal concentration (MBC) were determined on planktonic cells and crystal violet assays performed to assess the biofilm inhibition and disruption amount. Results: No inhibitory effect was observed on F. nucleatum biofilms (P > 0.05). Significant disruptive properties on A. actinomycetemcomitans at values lower than the MIC (3.90 µg/mL) was detected (P < 0.05). Shiitake mushroom oil had significant inhibitory and disruptive effects on P. gingivalis (P < 0.05). Furthermore, S. mutans and multispecies biofilms were inhibited at the same concentrations (15.60 -32.25 µg/mL). Conclusions: Shiitake essential oil is an effective anti-bacterial and anti-biofilm agent on important oral pathogens and has a possible therapeutic potential as an oral anti-biofilm agent.