Ozonation of natural surface water increases the concentration of oxygen-containing low molecular weight compounds. Many of these compounds support microbiological growth and as such are termed assimilable organic carbon (AOC). Phytoplankton can contribute substantially to the organic carbon load when surface water is used as source for drinking water treatment. We have investigated dissolved organic carbon (DOC) formation from the ozonation of a pure culture of Scenedesmus vacuolatus under defined laboratory conditions, using a combination of DOC fractionation, analysis of selected organic acids, aldehydes and ketones, and an AOC bioassay. Ozonation of algae caused a substantial increase in the concentration of DOC and AOC, notably nearly instantaneously upon exposure to ozone. As a result of ozone exposure the algal cells shrunk, without disintegrating entirely, suggesting that DOC from the cell cytoplasm leaked through compromised cell membranes. We have further illustrated that the specific composition of newly formed AOC (as concentration of organic acids, aldehydes and ketones) in ozonated lake water differed in the presence and absence of additional algal biomass. It is therefore conceivable that strategies for the removal of phytoplankton before pre-ozonation should be considered during the design of drinking water treatment installations, particularly when surface water is used.