Numerous studies have determined the effects of physical disturbance on periphyton, however, the substrata used have varied in size among studies. In this study we examined the influence of substratum size on the change in periphyton exposed to three levels of disturbance. Periphyton communities were established in a large greenhouse tank on square unglazed tiles that were either 2.5, 5, or 7.5 cm on a side. Following community development sets of tiles were randomly assigned to controlled disturbances removing 25%, 50%, or 75% of the community or an undisturbed control. Following disturbance, changes in periphyton density were associated with both disturbance treatment and tile size as was taxa richness. Experimental results and direct observation revealed that algal growth was most concentrated along the edge of the tile and progressively declined toward the center. Thus, substratum size influences colonization and pre-disturbance community structure, which then affects the extent of periphyton community change due to different levels of disturbance.