Since periphytic biofilm is an important source of food in lotic ecosystems, it is important to understand how key ecological factors affect the accrual and loss of algal biomass and sediment in the biofilm. We designed a field experiment to evaluate the effects of mesohabitat type (pools and riffles), grazing fish (control and exclusion), and substrate roughness (smooth and rough) on chlorophyll a, ash-free dry mass (AFDM), and total dry mass in a subtropical stream. Mesohabitat type did not influence the effect of grazers on periphyton. However, rough substrates accumulated more total dry mass in pools than in riffles, while smooth substrates accumulated similar amounts of total dry mass in both mesohabitats. The accrual of AFDMand chlorophyll a was greater on rough than on smooth substrates, regardless of mesohabitat. Treatments without fish accrued more total dry mass, AFDM, and chlorophyll a than treatments with fish, showing that fish play amajor role in this stream by removing sediment and algal biomass. These results suggest that habitat simplification in the scale of substrate roughness and loss of large grazers may impact the accrual and loss of algal biomass and sediment in lotic ecosystems.