Fusarium equiseti UMN-1 fungal strain isolated from soybean is selected as a potential oleaginous fungal strain for biodiesel generation. It possesses desirable features, such as high lipid content (up to 56%) and high fatty acid methyl ester (FAME) content (more than 98%) in total lipids, and also has the capability to produce cellulase. This research focused on the investigation of the characteristics of this strain and optimization of culture conditions to enhance lipid production. Impact of temperature, agitation, C/N ratio, medium composition, and carbon and nitrogen sources has been observed, and central composite design (CCD) has been applied to improve the lipid accumulation. The optimum range for temperature, agitation, C/N ratio, and carbon and nitrogen concentrations was discovered, and the CCD model with the optimized growth medium and growth conditions achieved a maximum lipid production of 3.89 g/L. This research on F. equiseti UMN-1 fungal strain is expected to improve the feasibility of using microbial lipids of F. equiseti UMN-1 strains as the source of biofuels.