AimsThe aim of this study was to isolate novel antibiofilm compounds produced by environmental bacteria. Methods and ResultsCell-free extracts were prepared from lawns of bacteria cultured on agar. A total of 126 bacteria isolated from soil, cave and river habitats were employed. Extracts were tested for their ability to inhibit Staphylococcus aureus biofilm in a 96-well microtitre plate assay. A total of 55/126 extracts (44%) significantly inhibited Staph.aureus biofilm. Seven extracts were selected for further analysis. The antibiofilm activities in all seven extracts exhibited unique patterns of molecular mass, chemical polarity, heat stability and spectrum of activity against Staph.aureus, Staphylococcus epidermidis and Pseudomonasfluorescens, suggesting that these seven antibiofilm activities were mediated by unique chemical compounds with different mechanisms of action. ConclusionsEnvironmental bacteria produce abundant and diverse antibiofilm compounds. Significance and Impact of the StudyScreening cell-free extracts is a useful method for identifying secreted compounds that regulate biofilm formation. Such compounds may represent a novel source of antibiofilm agents for technological development.