This study presents in detail the biochemical and bioinformatic insights into a fungal secondary metabolite identified as asperyellone. In brief, yellow color pigment asperyellone (AY) obtained from Aspergillus species strain AN01 was explored for free radical scavenging (H₂ O₂, NO, O₂⁻), reducing power and enzyme-inhibiting activities. Investigations were conducted on the molecular structure of AY from density functional theory (DFT) B3LYP/6-311G(d,p) using the GAUSSIAN 09W software, which explored the electronic spectrum, highest occupied molecular orbital (HOMO) lowest unoccupied molecular orbital (LUMO) energy gap and intra-molecular charge transfer (ICT). The molecular electrostatic potential (MEP), Mulliken atomic charges and various thermodynamic parameters were calculated accordingly. Furthermore, the enzyme inhibitory profiles of AY and its four analogues (asperenone, hydroasperyellone, CHEMBL1715716 and CHEMBL2152350) against eight targeted enzymes (HMGR, HNE, SQS, Tyrosinase, XO, LOX, MMP2 and MMP9) are detailed. Additionally, molecular physicochemical, bioactivity, ADMET and TOPKAT analyses were also carried out. The outcome of the present study provides useful information for the design of a potent and selective enzyme inhibitor from natural products (especially from microbial sources).