Chlamydomonas and other microalgae are capable of assimilating exogenous organic compounds. Additional carbon and energy sources affect different aspects of cell physiology. Simultaneous comparison of an auto- and mixotrophic batch of Chlamydomonas cultures indicates that acetate strongly stimulates the growth rate as well as the activity of respiration and photosynthesis in the exponential growth phase. In the middle of exponential growth, mixotrophy was characterized by a decrease in chlorophyll content that was followed by its elevation in the stationary phase. Chlamydomonas metabolomic profiles detected with GS-MS differed significantly as a result of trophic conditions. The maximum distinction was also observed in the middle of the exponential phase. The main feature of autotrophic cells is a higher carbohydrate content. Under the presence of acetate, cells demonstrated an intensive accumulation of fatty acids. This phenomenon is possibly related to the stimulation of lipid synthesis in the presence of additional sources of acetyl groups and by a higher content of carboxylates that were the result of elevated respiration. In the late stationary phase, mixotrophic cells exhibited a metabolomic shift towards autotrophy, probably due to acetate exhaustion. The importance of autotrophy at later stages of batch culture development correlated with chlorophyll accumulation in the early stationary phase. Thus, complicated trophic-induced metabolic and physiological alterations were revealed in Chlamydomonas cells during growth.