Aims The present work aims to explore a new oleaginous Fusarium isolate potential to accumulate lipids in its biomass from inexpensive substrates. In addition, impacts of carbon and nitrogen sources and their ratios on lipid production by the interested fungal isolate were also studied. Methods and Results Lipid was assayed by sulfo‐phospho‐vanillin colorimetric method. Among 11 Fusarium isolates obtained on potato dextrose agar from rhizosphereic soils, Fusarium RAS18 was selected as the highest producer that accumulates above 20% lipid. It was identified based on phenotypic characterization and the internal transcribed spacer sequence as Fusarium solani, that was recorded in the GenBank database under the accession number . The optimized lipid yield (34·5%) is obtained using glycerol (35 g l−1) and peptone (1·5 g l−1) as carbon and nitrogen sources respectively. The produced fatty acid methyl esters (biodiesel) is composed of linoleic acid (56·81%), palmitic acid (17·81%), oleic acid (11·81%) and stearic acid (11·12). The unsaturated fatty acids accounted for 69% and this is nearly similar to the plant oils commonly used in biodiesel production. Conclusions These findings suggest the applicability of F. solani RAS18 as a promising strain to accumulate lipids from glycerol as a feedstock for biodiesel production. Significance and Impact of the Study Fusarium solani RAS18 is a new oleaginous fungal isolate that is able to produce lipid (34·5%, g g−1) from glycerol. Glycerol is a cheap substrate and is formed as a byproduct from transesterification process and others industries. Thus, recyclation of glycerol for lipid production by micro‐organisms is an important point of economic view. Direct transesterification of the produced fatty acids indicated its similarity to the plant oil composition used in biodiesel production. So, F. solani RAS18 might be a potential lipid source as a feedstock for biodiesel production.