Gundelia species are among significant key medicinal plants extensively utilized in folk medicine of Middle Eastern countries. This study focused on researching the biopharmaceutical potency and bioactive compounds of Gundelia rosea seed. Hereby, traditional knowledge-based preparing methods (infusion and decoction) and ethanol-based lyophilized extracts obtained from Gundelia rosea seeds were assessed for (i) antioxidant capacities, (ii) enzyme inhibitory activities, (iii) HPLC-MS/MS and (iv) GC–MS studies. Phytochemical analysis revealed that ethanol extract which primarily compromised of mainly phenolics (4-Caffeoylquinic acid and luteolin hexoside) and several fatty acids (palmitic, stearic, oleic and linoleic acids), was superior to those of infusion and decoction extracts. Antioxidant activities findings revealed that ethanol extract contained a high level of total phenolics (55.3 mg Gallic acid Eq./g extract) and had high capacities of reducing (1683 μmol Fe2+ and 214.1 mg Trolox Eq./g extract for FRAP and CUPRAC respectively) and radical scavenging (ORAC: 2241.9 μmol, DPPH: 91.7 mg, ABTS: 141.2 mg Trolox Eq./g extract) and total antioxidant (Phosphomolybdenum: 1.39 mmol Trolox Eq./g extract) properties. The suppressive abilities of the extracts against selected isolated enzymes revealed that ethanol extract had pronounced levels of inhibitory activities against AChE (4.3 mg Galanthamine Eq.), BChE (3.4 mg Galanthamine Eq.), tyrosinase (120 mg Kojic acid Eq.), amylase (0.61 mmol Acarbose Eq.), glucosidase (11.91 mmol Acarbose Eq.) and lipase (53.4 μmol Orlistat Eq.) per gram extract. Findings obtained within this study confirmed the traditional utilization of Gundelia rosea and suggest its potential as a novel candidate of biopharmaceutical agents for public health problems.