Antibacterial and antifungal activities of Lithospermum ccinescens roots extract and acetylshikonin, isobutyrylshikonin — naphthoquinone derivatives isolated from them were investigated by measuring their minimal inhibitory concentrations (MIC). L. canescens extract was found to have strong antibacterial and antifungal activities against investigated Gram-positive bacteria: Staphylococcus aureus 1251, Enterococcus faecalis ATCC 8040, and yeast-like fungi strain Candida albicans PCM 1409 PZH and comparable to chloramphenicol and amphotericin B activities. However, the extract potency against Gram-negative bacteria such as Escherichia coli PZH 026 B6 and Pseudomonas aeruginosa S. 85/2 was several times lower. Acetylshikonin and isobutyrylshikonin showed strong antibiotic activity. The effect of these compounds on the S. aureus FDA 209P was similar as effect of chloramphenicol.