This study investigated the effect of patulin and penicillic acid, two known quorum-sensing inhibitors, and the common biocide ethylenediaminetetraacetic acid (EDTA) on the biofilm formation and auto-inducer (AI)-2 production of three isolates from dental unit water lines, Klebsiella sp., Bacillus subtilis and Bacillus cereus. Penicillic acid on its own had no effect on the biofilm formation of all isolates, whereas in combination with EDTA, it enhanced biofilm formation significantly in Klebsiella sp. and B. cereus. EDTA at concentrations greater than 10 mu M promoted biofilm formation in B. cereus and B. subtilis. Patulin was found to promote biofilm formation in B. cereus up to 25 mu M. A significant increase in biofilm formation was observed in B. cereus and B. subtilis at concentrations greater than 10 mu M of patulin when combined with EDTA. The Vibrio harveyi BB170 AI-2 bioassay showed a positive response for Klebsiella sp. AI-2 production with a maximum fold induction at the late exponential growth phase. Addition of glucose prolonged the AI-2 production phase considerably. No significant effect of patulin, penicillic acid alone as well as in combination with EDTA was observed on AI-2 production by Klebsiella sp. The findings have important implications for the design of biofilm prevention and eradication strategies.