Biochemical composition of microalgae, a promising feedstock for biodiesel production, can be modified under different nutritional modes. The objective of this study was to investigate the influence of autotrophy and mixotrophy on the growth and biochemical composition of Tetraselmis sp. KY114885. Time-course variations of lipids and starch were determined using gravimetric and colorimetric methods. After direct transesterification, lipid profile on days 12 and 20 of cultivation period was determined. The results revealed higher growth of microalgae in mixotrophic cultures containing 5 g L−1 glucose, whereas 7.5 g L−1 glucose suppressed growth. Mixotrophy enhanced the lipid and starch yields in a distinctive trend. The fatty acid composition changed with the different nutritional regimes and the length of growth in batch culture. Glucose supplementation elevated the portions of monounsaturated fatty acids, particularly oleic acid, and decreased the percentage of saturated and polyunsaturated fatty acids. The present study suggests that mixotrophic cultivation of Tetraselmis sp. KY114885 may be a feasible strategy to improve growth parameters and the biochemical composition of the alga for biofuel production. The results also shed light on carbon allocation in the algae grown under two different trophic modes.