Greek oregano is a well-known spice used worldwide in food, cosmetic, and pharmaceutical industries. The present study aimed to determine the effect of increasing nitrogen doses (0, 30, 60, 90, 120, and 150 kg ha−1) on polyphenol content, antioxidant activity, and bioaccessibility of Greek oregano cultivated in Poland. Additionally, the yield and content of active substances (essential oils and flavonoids) in oregano raw material during the first and second year from its establishment were analyzed to assess the effects of both nitrogen and growth season in moderate climate conditions. The application of different doses of nitrogen affected the plant height and number of stems per plant. Thus, in both the compared years, the plants were shorter and produced a lower number of stems in the control treatment, while treated plants showed increasing height and stem number with higher doses of nitrogen (up to 120 kg N ha−1). Consequently, the fresh weight and air-dry matter of the aboveground parts of single treated plants were enhanced by nitrogen application as compared to those of the control plants. Nitrogen application also increased the dry matter of whole and rubbed herb yields per area unit, exhibiting an optimum at 120 kg N ha−1, while the agronomic efficiency was the highest with nitrogen application at low doses. Nitrogen application also positively influenced essential oil yield, with an optimum at 90 kg N ha−1, through its favorable effects on herbage yield. However, the essential oil concentration systematically decreased (because of the dilution effect). Furthermore, simulated gastrointestinal digestion was used to determine the content of polyphenols and antioxidant potential of the in vitro bioaccessible fraction. The bioaccessibility of phenolic compounds ranged from 75.1%–72%; however, a slight negative effect of nitrogen application was observed on the bioaccessibility of phenolic compounds (especially at doses equal to or higher than 60 kg N ha−1).