Increasing environmental pollution is driving an increase in the production and use of biofuels. The cost price of biodiesel could be reduced by using low-quality oilseeds unfit for human consumption and by applying the simultaneous oil extraction and transesterification process, avoiding the oil pressure stage. The purpose of this study was to investigate the enzymatic biofuel production process (in situ) by using rapeseed with high oil acidity for simultaneous oil extraction and transesterification with a mixture of butanol and mineral diesel fuel. The investigation of the in situ process was performed using a mixture of butanol and mineral diesel and the most effective biocatalyst Lipozyme TL IM was selected. The novelty of this paper consists of the fact that mineral diesel was used as the oil extractant, and the amount chosen was such that, at the end, a mixture of fuel with a ratio 9:1 of mineral diesel to biodiesel was be produced. The experiments were carried out using ground rapeseeds under laboratory conditions. The efficiency of oil extraction was investigated by the FTIR spectrometry method, and the efficiency of transesterification was determined by the gas chromatography method. It was found that the optimal reaction duration was 7 h, reaction temperature was 40 °C, and lipase content was 6% (from the oil content in rapeseed). An oil extraction efficiency of 99.92 ± 0.04 (w/w) was observed at these conditions. A transesterification degree of 99.08 ± 0.08% (w/w) met with the requirements of the standards for biodiesel fuel. The physical and chemical properties of the produced fuel mixture met the requirements of the standards for mineral diesel and biodiesel; therefore, it can be used in diesel engines.