The rapid rise of resistance causes existing antibiotics to become dysfunctional. Therefore, search for new antimicrobial active ingredients has increased in recent years. In this study, flower extracts of Castanea sativa were examined for antimicrobial and anti-quorum sensing aspects. The antimicrobial properties of methanol, ethyl acetate, ethanol and hexane extracts of C. sativa against some gram-positive and gram-negative bacterial species, as well as yeasts (Candida albicans and Candida parapsilosis) were investigated by the agar well diffusion method. The minimum inhibition concentration (MIC) and minimum bactericidal concentrations (MBC) of C. sativa extracts were also determined. Chromobacterium violaceum ATCC 12472, C. violaceum 35352, C. violaceum VIR07 and C. violaceum CV026 indicator strains were used for determination of the quorum sensing inhibitions, and the Pseudomonas aeruginosa PAO1 strain was used for the swarming tests. Additionally, biofilm inhibition was detected by the spectrophotometric method using the P. aeruginosa PAO1 strain. Methanol, ethanol and ethyl acetate extracts of C. sativa was found to have high antibacterial and antifungal effects, while the methanol extract also had anti-quorum sensing, anti-swarming and biofilm inhibition effects, but no activity was found in the n-hexane extract. To the best of our knowledge this is the first report revealed that methanol extract obtained from C. sativa flowers induced anti-quorum sensing activities mainly inhibited the violacein production, swarming and biofilm formation. The present investigation provided evidence that the C. sativa flower extract maybe a potential source of antimicrobial agents. Therefore, much attention should be paid to C. sativa flower content, which could be used with high efficacy against microorganisms.