Aims: To evaluate the antimicrobial properties of flavonoid-rich fractions derived from bergamot peel, a byproduct from the Citrus fruit processing industry and the influence of enzymatic deglycosylation on their activity against different bacteria and yeast. Methods and Results: Bergamot ethanolic fractions were tested against Gram-negative bacteria (Escherichia coli, Pseudomonas putida, Salmonella enterica), Gram-positive bacteria (Listeria innocua, Bacillus subtilis, Staphylococcus aureus, Lactococcus lactis) and the yeast Saccharomyces cerevisiae. Bergamot fractions were found to be active against all the Gram-negative bacteria tested, and their antimicrobial potency increased after enzymatic deglycosylation. The minimum inhibitory concentrations of the fractions and the pure flavonoids, neohesperidin, hesperetin (aglycone), neoeriocitrin, eriodictyol (aglycone), naringin and naringenin (aglycone), were found to be in the range 200 to 800 mu g ml(-1). The interactions between three bergamot flavonoids were also evaluated. Conclusion: The enzyme preparation Pectinase 62L efficiently converted common glycosides into their aglycones from bergamot extracts, and this deglycosylation increased the antimicrobial potency of Citrus flavonoids. Pairwise combinations of eriodictyol, naringenin and hesperetin showed both synergistic and indifferent interactions that were dependent on the test indicator organism. Significance and Impact of the Study: Bergamot peel is a potential source of natural antimicrobials that are active against Gram-negative bacteria.