Antimicrobial peptides in the thionin family are known to have inhibitory activity against a wide range of microorganisms and tumor cells. Here, we represent the results of a study on thionins that were isolated from black cumin (Nigella sativa L.). N. sativa is a common species that is used as a medicinal plant. Ten novel thionins (NsW 1–10) were isolated from N. sativa seeds using acetic acid extraction followed by liquid chromatographic fractionating. The molecules obtained were assigned to the thionin family according to their primary structure. Four of the molecules were tested in the functional study. Activity against three clinical Aspergillus species was detected for three of the tested peptides. Activity against Candida albicans and Gram-positive bacteria was also shown. C. albicans cell death occurred by disruption of cell membrane integrity. This mode of action on the yeast cells was confirmed by studying the effect of peptides on the tumor cells in vitro. Upon incubation of the peptides with four different cancer cell lines (AsPC-1, Colo357, RD, Jukart), a dose-dependent cytotoxic effect was found. The recombinantpeptide NsW2 was obtained using heterologous expression in a prokaryotic model of the Escherichia coli Origami strain. The target peptide was obtained in association using a chimerical construction with the original E. coli protein thioredoxin, which provides normal peptide molecule folding. Insecticidal action of NsW 1-2 toward a mealworm (Tenebrio molitor) and a flesh fly (Sarcophaga carnaria) via direct injection to the hemolymph was detected. There was no cytotoxic effect toward the Sf9 insect cell line. Thus, there is likely another molecular mechanism of insectotoxicity that may be associated with neuromuscular transmission blocking or have specific activity on ion channels.