Fatty acid methyl esters (FAMEs), as a transformer insulating oil, and value-added glycerol derivatives were simultaneously synthesized from soybean oil by transesterification using Lipozyme 435 in dimethyl carbonate (DMC). The conversion of transformer insulating oil and glycerol derivatives reached 92% and 72%, respectively, under optimum conditions (DMC-to-oil molar ratio of 4.5:1 with 0.5 v/v % water and 15 wt. % Lipozyme 435 at 70 °C) in one-pot batch reactions. The purified transformer insulating oil possessed a dielectric breakdown voltage of 82.0 kV, which is sufficiently high for transformer insulation oil applications. Other properties such as density, dielectric breakdown voltage, and viscosity were comparable or superior to those of mineral oil, confirming that achieved material could be used as an alternative transformer insulating oil. Additionally, the glycerol was simultaneously converted into glycerol derivatives, which can be utilized as ingredients for cosmetics or monomers for bio-based plastics. This study clearly demonstrates that transformer insulating oil and value-added glycerol derivatives were simultaneously produced based on the zero-waste utilization of soybean oil.