Isohexenylnaphthazarins are commonly found in the root periderm of several Boraginaceous plants and are known for their broad range of biological activities. The work described herein concerns the biological activity of compounds from the roots of Echium plantagineum L. and Echium gaditanum Boiss (Boraginaceae) collected from field sites in southern Spain and Australia. Bioactivity was assessed using etiolated wheat coleoptile bioassay and in vitro growth inhibitory activity in HeLa and IGROV-1 cells. The quantification of four isohexenylnaphthazarins (shikonin/alkannin, deoxyshikonin/deoxyalkannin, acetylshikonin/acetylalkannin and dimethylacrylshikonin/dimethylacrylalkannin) was performed by LC-MS/MS using juglone as internal standard. Correlation coefficient values for the activities and concentrations of these four analytes were in the linear range and were greater than 0.99. Acetylshikonin/acetylalkannin and dimethylacrylshikonin/dimethylacrylalkannin were present in the highest concentrations in extracts of both species. The results reveal that greatest overall inhibition was observed in both bioassays with E. gaditanum extracts. Strong correlations between time of collection, sampling location and bioactivity were identified.