© 2021 by the authors. Licensee Italian Society for Horticultural Science (Società di Ortoflorofrutticoltura Italiana; SOI), Sesto Fiorentino (Firenze), Italy. This work is an open access article distributed under a Creative Commons Attribution-NonCommercial (CC BY NC) 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/).Oregano (Origanum vulgare L.) is a multi-purpose plant species with medicinal, culinary, and ornamental merit, which needs to be produced at an industrial scale. In the present research, effects of N-acetylated (10%) chitosan (CHT), 6-benzylaminopurine (BAP), and kinetin (KIN) on in vitro seed germination and shoot development in O. vulgare were studied in a concentration-dependent manner. The seed germination and shoot induction rates were mostly enhanced after CHT and KIN treatments, while BAP treatments reduced the rates. The chitosan treatment significantly triggered leaf production and shoot elongation better than the cytokinin treatments. The maximum shoot length (4.38±0.17 cm) and leaf number (17.71±0.69 leaves per shoot) were reached after chitosan treatment at 0.75 mg L-1, while shoot numbers were reduced after all chitosan and cytokinin treatments at low levels. However, shoot production was significantly enhanced in the presence of 2.0 and 4.0 mg L-1 KIN in the medium. Adventitious rooting without using auxins also occurred during the incubation period. The cluster analysis showed that the effects of 0.50 and 0.75 mg L-1 CHT treatments were better than BAP and KIN in stimulating shoot growth and leaf development in oregano. This study suggested that CHT might be used as a substitute for synthetic cytokinins to produce longer shoots and a high number of leaves in oregano propagation. Therefore, CHT might play a cytokinin-like role in plant tissue cultures. However, the effects of chitosan treatment might differ among species and according to its chemical structure.