Natural product is a well-known source of bioactive compounds. Herein, a steroidal compound stigmasta-7,22-diene-3-one (stigmastadienone) has been isolated from Isodon rugosus. The potency of isolated compound has been tested for several in-vitro targets. The acetyl and butyrylcholinesterase assays were performed using Ellman's procedure. For the in-vitro antidiabetic potential, α-glucosidase inhibitory assay was performed. Similarly, the cyclo and lipoxygenase pathways were studied to find its potential role in the management of inflammation and analgesia. The 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and hydrogen peroxide (H2O2) assays were performed for the antioxidant potentials. Docking studies were performed against acetylcholinesterase, cyclooxygenase and lipoxygenase targets. In anticholinesterase assays, stigmastadienone exhibited half-maximal inhibitory concentration (IC50) values of 13.52 and 11.53 μg/ml for acetyl and butyrylcholinesterase respectively. The observed IC50 values for that of galantamine were 6.07 and 4.42 μg/ml for acety and butyrylcholinesterase respectively. In inhibiting α-glucosidase enzyme, the compound showed mediocre IC50 of 109.40 μg/ml compared to the standard acarbose (7.60 μg/ml). The stigmastadienone proved to be an excellent inhibitor of cyclooxygenase 2 (COX-2) and 5-lipoxygenase (5-LOX) attaining IC50 values of 4.72 and 3.36 μg/ml respectively. The standard drugs IC50 values for COX-2 (celecoxib) and 5-LOX (montelukast) were 3.81 and 2.74 μg/ml respectively. The enzymatic activities of stigmastadienone were also supplemented with antioxidant results, specifically it was more dominant against DPPH and ABTS free radicals. Docking studies showed that only the carbonyl oxygen is able to form hydrogen bond interaction with the residues. In conclusions, the stigmastadienone has been isolated from Isodon rugosus for the first time. Moreover, the compound has been evaluated for several biochemical pathways which suggest its pharmacological role on the explored targets. Copyright © 2021 Elsevier Inc. All rights reserved.