This study was undertaken to evaluate the effect of surface roughness on the attachment and removal of Escherichia coli O157:H7 on selected fruit and metal surfaces. A new method to determine surface roughness was developed using confocal laser scanning microscopy (CLSM). A series of 2-D layered images were taken by CLSM optical slicing of the surfaces of Golden Delicious apples, navel oranges, avocadoes, and cantaloupes. The average roughness (R(a)) of the fruit surfaces was assessed by reconstructing a series of 2-D images into 3-D images. A cocktail of 5 E. coli O157:H7 strains were spot inoculated onto fruit skin surfaces with different R(a). The fruits were then treated with acidic electrolyzed water (AEW), peroxyacetic acid (POAA), and sterilized deionized water. Aluminum stubs with different R(a) values as a model system were also spot inoculated with E. coli O157:H7 and subjected to a sonication treatment. Test results indicated that there was a positive linear correlation between R(a) and adhesion rate of E. coli O157:H7, and a negative correlation between R(a) and the efficacy of inactivation by AEW and POAA, respectively, on fruit surfaces. A linear increase of residual bacteria population with increased surface roughness of aluminum stubs was also observed. The relationship between surface roughness and surface hydrophobicity was negative linear for the aluminum stubs, but was quadratic for the 4 fruits. The environmental scanning electron microscopy images showed that bacteria tended to attach to or be entrapped in the grooves or cavities of fruits, which provided protection to the cells against washing treatments.