Escherichia coliandEnterobacter cloacaeare major foodborne pathogens and can form challenging single/mixed biofilms. A recent study demonstrated that luteolin (LUT) exhibits antibacterial activities against some pathogens; however, the mechanisms underlying the effects of LUT on planktonic and biofilm bacteria have never been fully elucidated. This study aimed to determine the antibacterial activity and its mechanism of action againstE. coliandE. cloacae. Here, the antimicrobial mode of LUT was explored by evaluating alterations in both cell membrane integrity and cell morphology, and the antibiofilm activity of LUT was investigated using quantitative and qualitative assays. The results showed that minimal inhibitory concentration and minimum bactericidal concentration values of LUT againstE. coliwere 64 and 128 mu g/mL and 128 and 256 mu g/mL forE. cloacaemono- and dual-species, respectively. LUT impaired cell membrane integrity, as demonstrated by the remarkable increase in the number of membrane-damaged cells and definite variations in cell morphology. Moreover, LUT presented robust inhibitory effects on biofilm formation and the capacity to kill mono- and dual-species biofilm cells. Overall, these data show the potential benefit of using a natural antimicrobial and/or preservative in the food industry, LUT, to control mono- and mixed-species or biofilm-associated infections.