In the present study, the chemical composition of the volatile oil and methanolic extract from Ducrosia flabellifolia Boiss. was investigated. The antimicrobial, antioxidant, and anticancer activities of the methanolic extract from D. flabellifolia aerial parts were screened using experimental and computational approaches. Results have reported the identification of decanal (28.31%) and dodecanal (16.93%) as major compounds in the essential oil obtained through hydrodistillation. Farnesyl pyrophosphate, Methyl 7-desoxypurpurogallin-7-carboxylate trimethyl ether, Dihydro-Obliquin, Gummiferol, 2-Phenylaminoadenosine, and 2,4,6,8,10-dodecapentaenal, on the other hand, were the dominant compounds in the methanolic extract. Moreover, the tested extract was active against a large collection of bacteria and yeast strains with diameter of growth inhibition ranging from 6.67 ± 0.57 mm to 17.00 ± 1.73 mm, with bacteriostatic and fungicidal activities against almost all tested microorganisms. In addition, D. flabellifolia methanolic extract was dominated by phenolic compounds (33.85 ± 1.63 mg of gallic acid equivalent per gram of extract) and was able to trap DPPH• and ABTS•+ radicals with IC50 about 0.05 ± 0 mg/mL and 0.105 ± 0 mg/mL, respectively. The highest percentages of anticancer activity were recorded at 500 µg/mL for all cancer cell lines with IC50 about 240. 56 µg/mL (A-549), 202.94 µg/mL (HCT-116), and 154.44 µg/mL (MCF-7). The in-silico approach showed that D. flabellifolia identified compounds bound 1HD2, 2XCT, 2QZW, and 3LN1 with high affinities, which together with molecular interactions and the bond network satisfactorily explain the experimental results using antimicrobial, antioxidant, and anticancer assays. The obtained results highlighted the ethnopharmacological properties of the rare desertic D. flabellifolia plant species growing wild in Hail region (Saudi Arabia).