Technical Abstract: Confocal laser scanning microscopy (CLSM) was used in the reflection mode to characterize the surface texture (roughness) of sliced food surfaces. Sandpapers of grit size between 150 and 600 were used as the height reference to standardize the CLSM hardware settings. Sandpaper particle sizes were verified by scanning electron microscopy (SEM). The mean amplitude (in micrometer) of surface variation along randomly selected line segments of the scanned sandpaper images showed very close agreement between the measured result and the grit particle size (based on the United States Coated Abrasive Manufactures Institute, CAMI, standard). The established CLSM instrument settings were utilized to study the surface texture of mechanically-sliced food surfaces including cooked ham, salami and cheese. Sliced food surface texture parameters of Ra and Rs were evaluated by this method, where Ra and Rs are the roughness parameters on a line segment and on a region of interest basis, respectively. Values of the surface roughness of sliced ham, salami, and cheese were found to be in the range of the selected sandpaper surface roughness. The CLSM method may be useful for other surface texture measurements, and to investigate the impact of food surface texture on microbial adhesion or attachment, which might play a significant role in microbial transfer from one surface to another.