Two undescribed sesquiterpene coumarins (1–2) and eight known analogues (3–10) were isolated from the roots of Ferula samarkandica Korovin, growing in Uzbekistan. Their structures were established by comprehensive spectroscopic analysis including NMR, HRESIMS, UV and IR data. The absolute configurations were determined based on ECD calculations and confirmed by single-crystal X-ray diffraction analysis. Six compounds carrying 7′-carbonyl, 3′-carbonyl and 3′-acetoxy (4, 5, 6, 7, 9, 10) demonstrated anti-vitiligo potentials, in addition to five (4, 7, 8, 9, 10) with cytotoxic effects. Evaluation of anti-vitiligo activity in B16 cells revealed that compounds 4–7 and 9 exhibited promotion effects on melanin synthesis in B16 cells more potent than the positive control 8-methoxypsoralen (8-MOP), while 5, 7, 9 and 10 could also stimulate the tyrosinase activity superior to 8-MOP. Besides, compounds 4 and 7–10 also presented certain cytotoxic activities against HeLa, HT-29 and A549 cell lines, wherein 8 displayed a significant cytotoxicity against HT-29 cells with an IC50 value of 8.33 ± 0.34 μM.