Crude oils are highly complex mixtures containing many toxic compounds for organisms. While their level of toxicity in a marine environment depends on many parameters, one of the main factors is their composition. After oil spills, their compositions are significantly changed, so it changes the toxicity. In this study, different weathering processes such as evaporation, photooxidation, and biodegradation were applied to crude oil to understand how composition changed over time and how this affects its toxicity on phytoplankton. In laboratory settings, three distinct water-accommodated fraction samples of crude oil were prepared, unweathered, evaporated, and weathered and were exposed to phytoplankton communities at different dilution levels. After 3 days, evaporation reduced the crude oil concentration by 47%, and the concentration of the crude oil affected by photooxidation, biodegradation, and evaporation reduced by 81%. This study also showed that even though the weathering reduced the overall amount of crude oil substantially, its toxicity increased significantly. In the microcosm experiments, 7-day EC50 values of the unweathered oil, the evaporated oil and the weathered oil were 49.07, 21.09, and 7.16 µg/L, respectively. Different processes altered the crude oil composition, and weathered crude oil ended up with a higher fraction of high molecular weight (HMW) polycyclic aromatic hydrocarbons (PAHs). A promising relation between the increasing toxicity and HMW PAH fraction indicates that increasing the fraction of HMW PAHs might be one of the main reasons for the weathering process to cause higher crude oil toxicity. These results could be used as a diagnostic tool to estimate the extent of weathering and toxicity of crude oil after spills.