To prevent contamination of fresh produce with enteric pathogens, more insight into mechanisms that may influence the association of these pathogens with fresh produce is needed. In this study, Escherichia coli O157:H7 and Salmonella were chosen as model pathogens, and fresh cut iceberg lettuce was chosen as a model fresh produce type. The morphological structure of iceberg lettuce leaves (stomatal density and length of cell margins per leaf area) was quantified by means of leaf peels and light microscopy of leaves at different stages of development (outer, middle, and inner leaves of the crop) on both leaf sides (abaxial and adxial) and in three leaf regions (top, center, and bottom). The morphology of the top region of the leaves was distinctly different from that of the center and base, with a significantly higher stomatal density (up to five times more stomata), different cell shape, and longer cell margins (two to three times longer). Morphological differences between the same regions of the leaves at different stages of development were smaller or nonsignificant. An attachment assay with two attenuated E. coli O157:H7 strains (84-24h11-GFP and BRMSID 188 GFP) and two Salmonella strains (serovars Thompson and Typhimurium) was performed on different regions of the middle leaves. Our results confirmed earlier reports that these pathogens have a higher affinity for the base of the lettuce leaf than the top. Differences of up to 2.12 log CFU/g were seen (E. coli O157:H7 86-24h11GFP). Intermediate attachment occurred in the central region. The higher incidence of preferential bacterial attachment sites such as stomata and cell margins or grooves could not explain the differences observed in the association of the tested pathogens with different regions of iceberg lettuce leaves.