Microalgae are a promising alternative source of PUFAs, with Chlorella being one of the few microalgae widely available in the nutraceuticals market authorized for human consumption. This study explores the fatty acid (FA) profiles of nine C. sorokiniana biomass feedstocks produced under different combinations of light intensity and nitrogen and inorganic carbon loadings, derived via a Taguchi L9 (33−1) orthogonal array design. Additionally, the nutritional and medical value of Chlorella lipids using several nutritional indices is assessed. PUFAs were maximized under low light intensity and high nitrogen loading; however, these conditions favored the overaccumulation of omega-6 FAs. For omega-3 FA maximization, high light intensity must be applied, suggesting that high illumination induces the desaturation of linoleic acid to alpha-linolenic acid (ALA) in C. sorokiniana cells. Additionally, nitrogen-depleted conditions significantly downgraded its quality due to the overaccumulation of saturated FAs. Despite lacking EPA and DHA, C. sorokiniana lipids are an excellent source of ALA, surpassing concentrations met in plant-based oils. Thus, Chlorella lipids can be included in humans’ diet, satisfying daily ALA requirements; however, accurately labeling the FA profile of Chlorella products, prior to any nutritional claims, is indispensable, considering the sizeable variations in their profile under the impact of cultivation conditions.