|Microalgae have been recently cultivated using resources from wastewater treatment plants, including nutrients, CO2, and heat. In the present study, we focused on the natural cultivation of total microalgae rather than specific microalgae, for which culture conditions and the cultivation environment are difficult to prepare. Natural microalgal cultivation systems using 380-L raceway tanks were operated outdoors for 8 months and the effects of the culture substrate were investigated. The cultivation substrate was the primary effluent with or without excess sludge. The results showed that when diluted excess sludge was added to the substrate, microalgal biomass increased more than when the substrate contained only primary effluent. Additionally, the wastewater suspended matter and water quality were removed, reaching low levels. Microalgal culture systems using excess sludge showed higher organic acid content, higher biochemical methane potential, and higher efficiency in producing more microalgal biomass than wastewater treatment plant systems that did not use excess sludge.