Scabiosa columbaria is a plant traditionally used to treat skin ailments, such as scabies, wound bruises, sores and hyperpigmentation. To find a novel skin depigmenting agent, the present study was investigated to determine the possible anti-melanogenesis, antioxidant and anti-tyrosinase effects of methanol extract of S. columbaria leaves. Cytotoxicity towards human dermal fibroblast (MRHF) cells was assessed using the live-cell fluorescence imaging microscopy. The inhibitory effects of the extract on tyrosinase, collagenase and melanin synthesis were also investigated using standard in vitro method, while ferric reducing power (FRAP) was used to determine the antioxidant potential of the plant extract. The effect of the extract on collagen content in MRHF cells was also investigated. The plant extract displayed no meaningful cytotoxicity towards MRHF cells and no significant cell death was recorded at all the tested concentrations. The extract (25−100 µg/mL) effectively decreased melanin content in B16F10 (mouse melanoma) cells with moderate inhibition of tyrosinase enzyme in a dose-dependent manner. However, the extract also demonstrated no significant effect on collagenase and collagen content in MRHF cells, but showed strong antioxidant activity at the concentrations tested. The results suggest that S. columbaria could be a promising candidate in the treatment of skin hyperpigmentation disorders