Brassicaceae family provides several crops which are worldwide known for their interesting phytochemical profiles, especially in terms of content of glucosinolates. These secondary metabolites show several beneficial effects toward consumers’ health, and several studies have been conducted to identify cultivation factors affecting their content in crops. One of the agronomic practices which is attracting growing interest is the organic one, which consists in avoiding the use of mineral fertilizers as well as pesticides. The aim of this study is to define the metabolic profile of Brassica oleracea (var. italica) and to compare the samples grown using organic and conventional fertilization methods. The hydroalcoholic and organic extracts of the samples have been analyzed by NMR spectroscopy. Forty-seven metabolites belonging to the categories of organic acids, amino acids, carbohydrates, fatty acids, sterols, and other molecules have been identified. Thirty-seven metabolites have been quantified. Univariate and multivariate PCA analyses allowed to observe that the organic practice influenced the nitrogen transport, the carbohydrate metabolism, the glucosinolate content and the phenylpropanoid pathway in B. oleracea (var. italica).