Oleaginous fungi accumulate a high amount of lipids in their dry biomass and have attracted more attention, due to their broad potential in biotechnological applications. The present study aimed to isolate and evaluate efficient oleaginous endophytic fungi from selected plants, followed by optimizing the potential strain's growth parameters. A total of 51 morphologically distinct fungal isolates were isolated and examined for their oleaginous character. From the preliminary screening and fermentation assay, 13 fungal isolates were identified based on their high lipid with biomass or polyunsaturated fatty acids in their fatty acid profile. During our screening process, the fungal isolate identified as Gilbertella persicaria DSR1 was found to have desirable features, such as the highest cell dry biomass (12.7 g L−1) and lipid content (34.5% of biomass) with 7% γ-linolenic acid (GLA) of total fatty acids. The least investigated isolated oleaginous strain DSR1 was subjected to optimization of various growth parameters for exploiting its full lipid accumulation potential. Under optimized conditions in the fermentor, the strain DSR1 produced 14 ± 0.3 g L−1 dry cell biomass with 42 ± 1.4% total lipid content. The major fatty acid composition of lipid confirmed by GC analysis was palmitic acid (14.7%), stearic acid (10.6%), oleic acid (37.4%), linoleic acid (20.6%), γ-linolenic acid (13.4%). This is the first report showing the oleaginous nature of G. persicaria and this isolate may be further exploited as a potential candidate for biodiesel and GLA production in larger-scale applications.