Exposure experiments were conducted to determine the effects of an oil spill on a periphytic microbial community with the chemically enhanced water-accommodated fraction (CEWAF) of Iranian Heavy Crude oil in a microcosm containing artificial benthic substrates. Bacteria and heterotrophic nano-flagellates (HNF) grew well in all the treatments, except for the 100 % CEWAF exposure. However, periphyton did not adapt to concentrations of CEWAF ≥ 20 %. Among the periphyton, Cylindrotheca spp. dominated under treatment conditions, and the response of Cylindrotheca spp. to CEWAF (i.e., particularly 10 %) closely followed the changes in chlorophyll a concentration. The concentrations of petroleum hydrocarbons from 10 % and 20 % CEWAF seemed to have a negative effect on periphyton and a growth-promoting effect on bacteria and HNF, respectively.