Recent discoveries have shown that brominated furanones from marine red alga have remarkable activities against the colonization of bacteria and inhibit a wide spectrum of bacterial multicellular behaviors. To understand the mechanism of inhibition, it is important to study the structural effects on the activities of furanones. In this study, eleven new brominated furanones with systematic changes in the structure were synthesized so that their effects on microbial biofilm formation can be rigorously correlated to their structures. Highly pure regioisomers of brominated furanones were obtained (>98%) with optimized synthesis condition. The brominated furanones were tested for their effects on Escherichia coli biofilm formation on 316L stainless steel surfaces. E. coli was labeled with green florescence protein and its biofilms were studied with confocal laser scanning microscopy and the COMSTAT software. Four furanones were found to have statistically significant inhibition on E. coli biofilm formation in the order of 11 ≈ 8a ≈ 9 >12 at the concentration (60 mg/mL) nontoxic to the cells. Furanones 10 and 11 at 10 μg/mL (nontoxic to cell growth) were also found to reduce biofilm formation of Pseudomonas aeruginosa PAO1 in a 96-well plate by 85% and 31%, respectively, while 8a and 9 did not exhibit any significant inhibition at this concentration. Interestingly, furanones were found to inhibit the growth of Candida albicans; e.g., no growth was obtained for 24 hours in the presence of 2.5 μg/mL furanone 10. Thus, furanones may also control fungal biofilms. This and future work will help elucidate the mechanism of inhibition and design better antagonists.