These are datasets on benthic diatom in a three-factor split-split-plot field experiment consisting of the following hierarchical treatments: (i) mesohabitat (pools and riffles, five replicates for each level); (ii) grazer occurrence (presence or absence of fish, 10 replicates for each level); and (iii) substratum roughness (smooth and rough artificial substrata, 20 replicates for each level). In addition to the datasets, we also provide the R scripts.