[Objective] The aim of this study was to investigate the effects of antimicrobial compounds of Kluyveromyces marxianus (K2 and K8) on wild pathogenic Escherichia coli and its cell surface characteristics. [Methods] K2 and K8 were extracted by ethyl acetate, and the inhibition zones of K2 and K8 against E. coli O8 were determined by Oxford cup method. The organic acids were determined by HPLC, and the concentrations of killer toxin were determined by enhanced BCA Protein Assay Kit. The minimum inhibition concentration (MIC) and the minimum bactericidal concentration (MBC) were determined by broth dilution method, the effects of K2 and K8 on the growth curve of E. coli O8 were determined by turbidimetry. Moreover, the effects of K2 and K8 on the hydrophobicity of the E. coli O8 cell surface was determined using the microbial adhesion to solvents method, and the permeation of E. coli O8 cell membrane were determined by measuring the release of β-galactosidase activity into the culture medium using ONPG as a substrate. [Results] The aqueous phases of pH 2.0 and pH 8.0 had higher inhibition zones, they were dried for 48 h by freeze-drying, then, K2 and K8 were obtained, the main components were propanoic acid and some other organic acids, and killer toxins. The MICs of K2 and K8 were 0.025 g/mL and 0.100 g/mL, respectively. The MBCs of K2 and K8 were 0.100 g/mL and 0.200 g/mL, respectively. The growth curve of E. coli O8 was S-shape. It changed obviously after adding K2 and K8. E. coli O8 was basic character, and had a hydrophilic surface. The hydrophobicity increased after adding K2 and K8. In addition, the release of the β-galactosidase in permeation of E. coli O8 was promoted gradually by K2 and K8, and it also caused membrane lesions allowing ONPG uptake into cells. These two factors resulted in the increasing permeation. K2 was better than K8. [Conclusion] K2 and K8 could inhibit the growth of pathogenic E. coli O8 and influence its cell surface characteristics.