BACKGROUND Greek oregano is a culinary and medicinal herb native to the Mediterranean region; however nowadays it is cultivated in many regions all over the world. It is commonly used as a spice for flavoring food products and in various traditional medicine applications. This study investigated the effect of nitrogen fertilization at 0–150 kg N ha−1 on the potential bioaccessibility and activity of Greek oregano compounds. For this purpose the total phenolic content, rosmarinic acid content, and antioxidant activities of the raw material, as well as digested fractions, were determined. RESULTS Nitrogen fertilization had a negative influence on the phenolic content and antioxidant activity of raw material; however, its effect on the potential bioaccessibility varied depending on the dose. The highest potential bioaccessibility and activity was determined for plants fertilized with 30 kg N ha−1. For the gastric and intestinal phases of digestion, the potential bioaccessibility percentages were 39.5% and 29.6% for total phenolics, 53.1% and 11.2% for rosmarinic acid content, 45.2% and 44.4% for antiradical activity against 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 39.2% and 27.2% for antiradical activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 50.2% and 23.4% for reducing power, respectively. CONCLUSION The results showed that nitrogen fertilization is an important factor determining the in vitro bioaccessibility of Greek oregano compounds. Furthermore, the nutraceutical potential of herbs, in term of the bioaccessibility of bioactive compounds, may be optimized during plant cultivation by applying an accurate nitrogen level. © 2020 Society of Chemical Industry