Different environmental factors including agitation can affect the growth of microalgae and fatty acid composition. It is necessary to agitate the culture to ensure that each cell received enough amount of nutrition, air, and light exposure. This study aimed to analyze the growth and fatty acid composition of Acutodesmus obliquus Q2-12E cultivated with molecular diffusion (no agitation) and shaking and compare those with a previous study employing the aeration method. The results showed that in comparison to molecular diffusion, agitation by shaking and aeration on the culture significantly increased the specific growth rate. Culture under aeration was revealed to exhibit the fastest growth rate (0.1444 day-1) compared to shaking (0.0951 day-1) and molecular diffusion (0.0614 day-1). The lipid obtained using modified Folch’s method was esterified and analyzed using gas chromatography with a flame-ionization detector (GC-FID) with heptadecanoic acid (C17:0) as the internal standard. The analyzed result shows that the major fatty acids produced both by molecular diffusion and shaking method were monounsaturated fatty acids which were 39.5% and 46%, respectively. While the major fatty acid produced by the aeration method was polyunsaturated fatty acid (38.6%). Molecular diffusion and shaking could not produce eicosatetraenoic acid (C20:4) which was detected with the aeration method. This study demonstrated that the same microalga strain exhibited a different growth and fatty acid composition depending on the agitation methods.