The purpose of the investigation was the assessment of the effect of fertilization with different form and dose of nitrogen as well as harvest term on yield, chlorophyll, carotenoids and polyphenols content, and antioxidant activity of stinging nettle (Urtica dioica L.) leaves. The lowest total yield obtained from three-year-lasting plantation resulted from preplant fertilization with lime saltpeter. The highest yield of raw material was provided by nitrogen fertilization in the dose of 150 kg N/ha. Total content of polyphenols in nettle leaves collected in May amounted, counted over gallic acid, 7.13–9.46 mg∙g-1 d.m. and was by 40–50% higher than that assayed in July. Chlorophylls (7.72–9.63 mg∙g-1 d.m.) and carotenoids (0.74–0.96 mg∙g-1 d.m.) content was lower in May by 20–40% than the one in nettle leaves collected in July. In the course of effect of nitrogen preplant fertilization plants accumulated higher amounts of polyphenols when treated with N saltpeter form. Increased nitrogen dose from 50 to 150 kg N∙ha-1 contributed to the increase in chlorophyll and carotenoids content in nettle leaves and, to a lower degree, to differentiating polyphenols level. Harvest term did affect antioxidant activity of raw material more significantly than nitrogen form and dose used in the experiment. Raw material collected in May characterized higher antioxidant activity measured according to FRAP and ABTS.