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Ziziphora clinopodioides L. is a valuable medicinal and aromatic plant; however, its special requirements for proper growth and enhanced secondary metabolite composition have limited its production and availability. The lack of appropriate fertilizer dosage recommendations is one of these limiting factors for ex situ conservation and large-scale cultivation. This study investigated the effects of nitrogen (N) fertilization on Z. clinopodioides in both open field and unheated greenhouse conditions. The determined research factor was N dosage (0, 40, 80, and 120 kg N ha−1 in the field) and (0, 200, 400, and 600 mg N pot−1 in the greenhouse). It was found that Z. clinopodioides plants could grow successfully outside their natural habitat with sufficient N fertilization yielding a satisfactory amount of metabolites per plantation area. Specifically, among the tested dosages, the 120 kg N ha−1 (which showed no significant difference from 80 kg N ha−1) and 400 mg N pot−1 fertilizers increased the aboveground dry and fresh biomass, essential oil (EO) content, and yield. Nitrogen fertilization showed a direct correlation with menthone, neo-menthol, menthol, pulegone, E-ocimenone, and spathulenol, but a negative correlation with α-pinene, β-pinene, limonene, 1,8-cineol, trans-pulegol, and iso-menthone. This study is the first to evaluate the effects of N fertilization on the genus Ziziphora. The results indicate that utilizing N fertilizer at a moderate rate (80 kg N ha−1 in the field and 400 mg N pot−1 in the greenhouse) can improve the agronomic traits and EO of Z. clinopodioides.