As an opportunistic pathogen, Escherichia coli (E. coli) forms biofilm that increases the virulence of bacteria and antibiotic resistance, posing a serious threat to human and animal health. Recently, ginkgetin (Gin) has been discovered to have antiinflammatory, antioxidant, and antitumor properties. In the present study, we evaluated the antibiofilm and antibacterial synergist of Gin against E. coli. Additionally, Alamar Blue assay combined with confocal laser scanning microscope (CLSM) and crystal violet (CV) staining was used to evaluate the effect of antibiofilm and antibacterial synergist against E. coli. Results showed that Gin reduces biofilm formation, exopolysaccharide (EPS) production, and motility against E. coli without limiting its growth and metabolic activity. Furthermore, we identified the inhibitory effect of Gin on AI-2 signaling molecule production, which showed apparent anti-quorum sensing (QS) properties. The qRT-PCR also indicated that Gin reduced the transcription of curli-related genes (csgA, csgD), flagella-formation genes (flhC, flhD, fliC, fliM), and QS-related genes (luxS, lsrB, lsrK, lsrR). Moreover, Gin showed obvious antibacterial synergism to overcome antibiotic resistance in E. coli with marketed antibiotics, including gentamicin, colistin B, and colistin E. These results suggested the potent antibiofilm and novel antibacterial synergist effect of Gin for treating E. coli infections.