The development of innovative pharmacological formulations for the treatment and prevention of various major diseases, including cancer, diabetes, and glaucoma, has been facilitated by some natural compounds. This study tested the inhibitory effects of isofraxidin on acetylcholinesterase, α-glycosidase, and butyrylcholinesterase enzymes, as well as human carbonic anhydrase I and II (hCA I and II) isoenzymes. Esterase activity was used to gauge Isofraxidin's ability to inhibit CA (in vitro). For the isoenzymes hCA I and hCA II, the half maximal inhibitory concentration (IC50) values of isofraxidin were determined to be 67.61 and 52.42 nM, respectively. At the same manner, inhibition constant (Ki) values were determined as 12.58±0.50 and 4.41±0.35 nM, respectively. Then, IC50 value of compound for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were calculated as 18.50 and 10.75 nM, respectively. On the other hand, IC50 and Ki values of α-glycosidase were determined as 55.16 and 56.81±2.30 nM, respectively. Additionally, the antioxidant properties of isofraxidin were investigated using techniques like 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 1,1-Diphenyl-2-picrylhydrazyl (DPPH), N,N-dimethyl-ρ-phenylenediamine (DMPD), cupric ions (Cu2+) reducing antioxidant capacity (CUPRAC), and iron reduction procedures. Following the graphing of the antioxidant results, IC50 values were determined. As a result, the natural phenolic molecule showed strong profiles of inhibition profile. We therefore think that these findings may pave the way for novel therapeutic development for the management of some major illnesses. Several plant-based natural substances and extracts have gained attention in recent years as potential inhibitors of α-glycosidase enzyme. The activities of Isofraxidin molecule with various enzyme proteins were compared. Finally, ADME/T analysis was performed to predict the movements of Isofraxidin molecules in human metabolism.