The phytochemical investigation of the n-hexane extract from callus and cell suspension culture of Arnebia euchroma (Royle) Jonst. resulted in the isolation of nine isohexenylnaphthazarins: deoxyalkannin (1), alkannin (2), acetylalkannin (3), isobutyrylalkannin (4), β-hydroxyisovalerylalkannin (5), 2''-(S)-α-methylbutyrylalkannin (6), propionylalkannin (7), teracrylalkannin (8) and acetylshikonin (9). Their structures were determined by MS and NMR spectroscopy. Pigments 2–8 are isolated for the first time from Arnebia in vitro cultures, 4 and 7 are reported in the present work as novel metabolites within the Arnebia genus, while 9 is a known constituent of both natural roots and in vitro cultures of A. euchroma. Moreover, methyl jasmonate and 1-monoglyceryl olate, palmitate and stearate are reported for the first time within the Boraginaceae family. The antimicrobial and cytotoxic activities of all isolated pigment compounds were tested, revealing a very interesting profile.