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OBJECTIVE To determine the activity of alpha-mangostin on preformed bacterial-fungal multi-species biofilms in vitro, and to ascertain the impact on metabolic activity, biofilm structure and viability. DESIGN Inhibitory concentrations (ICs) for alpha-mangostin against planktonic cultures of Candida albicans, Enterococcus faecalis, Lactobacillus rhamnosus, and Streptococcus gordonii were determined using a standard broth microdilution method. Single and multi-species (all species 1:1:1:1) biofilms were grown on polystyrene coverslips in Roswell Park Memorial Institute Medium for 48 h. The biofilms were then exposed to 0.2% (w/v) alpha-mangostin for 24 h. These concentrations were selected based on pilot experiments and the solubility of these compounds. 2% (v/v) chlorhexidine was used as a positive control and Roswell Park Memorial Institute Medium as a negative control. The metabolic activity of the biofilms after exposure was measured using metabolic (XTT) assays. Biofilms were visualised and quantified using fluorescent BacLight™ LIVE/DEAD staining. The biofilms were assessed for cell viability by culture and colony counting (CFU/mL). RESULTS 8 mg/L of alpha-mangostin was cidal against planktonic bacteria and 1000 mg/L for Candida. Alpha-mangostin was most active against L. rhamonosus biofilms and least active against C. albicans biofilm (metabolism inhibited by 99% and 78%, respectively). Alpha-mangostin exposure reduced the number of viable cells in the biofilms. CONCLUSION Alpha-mangostin inhibited the metabolic activity of bacterial-fungal biofilms effectively. The anti-biofilm activity of alpha-mangostin was comparable to chlorhexidine and thus has potential as a novel agent for endodontic therapy.