Microalgal lipids are promising sustainable feedstocks for high-value functional ingredients. However, the influence of cultivation-driven lipid composition on enzymatic conversion remains poorly understood. This study integrated cultivation strategy and enzymatic upgrading to tailor Neochloris oleoabundans lipids for mono- and diacylglycerol (MAG and DAG) production. Heterotrophic cultivation achieved a maximum dry biomass concentration of 2.78 ± 0.14 g L−1, whereas autotrophic cultivation reached 0.39 ± 0.01 g L−1, confirming the superior biomass productivity of heterotrophic metabolism. Lipid fractions obtained under both trophic conditions were characterized and subjected to glycerolysis catalyzed by Novozym 435 under a 5:1 glycerol-to-oil ratio for 16 h. Heterotrophic oils, characterized by triacylglycerol-rich and low-free fatty acid (FFA) profiles, achieved higher MAG + DAG conversion (45%), while autotrophic oils reached 43% conversion despite elevated FFAs and polar lipids. The presence of FFAs, pigments, and phospholipids in nonrefined microalgal oils influenced catalytic behavior, reducing conversion efficiency and favoring competing esterification and hydrolysis pathways. These findings demonstrate that substrate purity, acylglycerol distribution, and cultivation-specific lipid architecture strongly affect lipase performance, highlighting oil refining and cultivation optimization as key strategies for improving sustainable MAG and DAG production.