“Shilajit” is a panacea in Ayurveda, the Indian traditional system of medicine. The major bioactives of “shilajit” have been identified as dibenzo-α-pyrones (DBPs), its oligomers and aminoacyl conjugated derivatives. These bioactive compounds play a crucial role in energy metabolism in all animal cells including those of man. 3-hydroxydibenzo-α-pyrone (3-OH-DBP), a key DBP component of “shilajit” is converted, among other products, to another active DBP derivative, viz. 3,8-hydroxydibenzo-α-pyrone, 3,8(OH)2-DBP, in vivo, when its precursor is ingested. 3,8(OH)2-DBP is then involved in energy synthesis in the mitochondria in the reduction and stabilization of coenzyme Q10 in the electron transport chain. As the chemical synthesis of 3,8(OH)2-DBP is a complex, multi-step process and economically not readily viable, we envisioned the development of a process using microorganisms for bioconversion of 3-OH-DBP to 3,8(OH)2-DBP. In this study, the biotransformation of 3-OH-DBP is achieved using Aspergillus niger, which was involved in the humification process on sedimentary rocks leading to “shilajit” formation. A 60% bioconversion of 3-OH-DBP to 3,8(OH)2-DBP and to its aminoacyl derivatives was achieved. The products were characterized and estimated by high performance liquid chromatography (HPLC), high performance flash chromatography (HPFC) and gas chromatography-mass spectrometry (GC-MS) analyses. Among the Aspergillus species isolated and identified from native “shilajit”, A. niger was found to be the most efficient for this bioconversion.