Plant sterols are well-known for their ability to reduce low density lipoprotein (LDL)-cholesterol and promote cardiovascular health, as well as anti-inflammatory, anti-atherogenicity, anti-cancer and anti-oxidative activities and protection against nervous system disorders. As considerable amounts of phytosterols are present in microalgae, we screened out of hundreds of Australian isolates, ten microalgal species for sterol profiles and total sterol content. The top three sterol producers at 0.4–2.6% dry weight were Pavlova lutheri, Tetraselmis sp. M8 and Nannochloropsis sp. BR2. To further increase sterol accumulation, the highest sterol producer, P. lutheri was subjected to nutrient and salinity changes. Although no significant immediate effects were observed for altered total sterol contents, individual sterols varied and, significantly higher total sterol amounts (up to a 2-fold increase) were found after prolonged cultivation. Total sterol accumulation of 5.1% dry weight was achieved in P. lutheri. Combined with the high areal productivity, commercial phytosterol production from microalgae could be considered.