In this paper, the synthesis of benzothiazole and thiourea derivatives with potential cytostatic activity is performed. Targeted N-acylthioureas (2a–f, 4a - i, 5a–i) were prepared by one-pot method from acyl chloride, via acyl thiocyanate intermediate and their addition to 2-aminobenzothiazole and 2-(4-aminophenyl)benzothiazole heterocycles. 2-Aminobenzothiazoles and 2-(4-aminophenyl)benzothiazoles substitued in position 6 were used for the synthesis. The 2-aminobenzothiazole (1a–f) derivatives required for the synthesis of N-acylthiourea were obtained by reaction of the appropriately substituted aniline with potassium thiocyanate in acetic acid, while 2-(4-aminophenyl)benzothiazole (3) was prepared by condensation of 2-aminothiophenol with 4-aminobenzoic acid in polyphosphoric acid (PPA) at 220°C. N-Acylthioureas were isolated and purified by recrystallization and column chromatography. In the synthesis of 2-aminobenzothiazole derivatives of acyl thioureas (2a–f) the corresponding amides (2a*–f*) were formed as by-products, while the synthesis of 2-(4-aminophenyl)benzothiazole derivatives of acyl thioureas (4a–i) and 6-methyl-2-(4-aminophenyl)benzothiazole derivatives of acyl thioureas (5a–i) gave only the desired products. 2-Aminobenzothiazole acyl-thioureas (2a–f) were obtained in lower yields (4 - 59%) compared to 2-(4-aminophenyl)benzothiazole acyl-thioureas (5a - i) (52 – 88%) and 6-methyl-2-(4-aminophenyl)benzothiazole acyl thioureas (6a–i) (22 – 89%). The structures of all compounds were confirmed by 1H and 13C NMR spectroscopy.