The cyclohexane (Ch) extracts of the roots of five Greek endemic Boraginaceae plants, Onosma kaheirei Teppner, 0. graeca Boiss., 0. erecta Sibth. & Sm., Alkanna sfikasiana Kit Tan, Vold and Strid and Cynoglossum columnae Ten, were investigated for the presence of alkannin/shikonin-related compounds. All species,s except C. columnae and 0. erecta, were found to contain this type of compounds. Seven compounds were obtained after several chromatographic separations from the Ch extracts of the investigated plants: deoxyalkannin (1), 2"-(S)-α-methylbutyrylalkannin (2), isobutyrylalkannin (3), propionylalkannin (4), acetylalkannin (5), β-hydroxyisovalerylalkannin (6), and β,β-dimethylacrylalkannin (7). All structures were identified by ID 1H-/¹³C- and 2D NMR spectroscopy, assisted also by ESI-MS. The extracts and the isolated compounds exhibiting an interesting antimicrobial profile when evaluated for their antimicrobial activity against six Gram-positive and -negative bacteria and three human pathogenic fungi.