Cumulative reported evidence has indicated that renewable feedstocks are a promising alternative source to fossil platforms for the production of fuels and chemicals. In that regard, the development of new, highly active, selective, and easy to recover and reuse catalysts for biomass conversions is urgently needed. The combination of enzymatic and inorganic heterogeneous catalysis generates an unprecedented platform that combines the advantages of both, the catalytic efficiency and selectivity of enzymes with the ordered structure, high porosity, mechanical, thermal, and chemical resistance of mesoporous materials to obtain enzymatic heterogeneous catalysts. Enzymatic mineralization with an organic silicon precursor (biosilicification) is a promising and emerging approach for the generation of solid hybrid biocatalysts with exceptional stability under severe use conditions. Herein, we assessed the putative advantages of the biosilicification technology for developing an improved efficient, and stable biocatalyst for sustainable biofuel production. A series of solid enzymatic catalysts denominated LOBE (low ordered biosilicified enzyme) were synthesized from Pseudomonas fluorescens lipase and tetraethyl orthosilicate. The microscopic structure and physicochemical properties characterization revealed that the enzyme formed aggregates that were contained in the heart of silicon- covered micelles, providing active sites with the ability to process different raw materials (commercial sunflower and soybean oils, Jatropha excisa oil, waste frying oil, acid oil from soybean soapstock, and pork fat) to produce first- and second-generation biodiesel. Ester content ranged from 81 to 93% wt depending on the raw material used for biodiesel synthesis. A heterogeneous enzymatic biocatalyst, LOBE4, for efficient biodiesel production was successfully developed in a single-step synthesis reaction using biosilicification technology. LOBE4 showed to be highly efficient in converting refined, non-edible, and residual oils (with high water and free fatty acid contents) and ethanol into biodiesel. Thus, LOBE4 emerges as a promising tool to produce second-generation biofuels, with significant implications for establishing a circular economy and reducing the carbon footprint.