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As part of a project aimed at investigating the Matthiola taxa endemic to Sicily (Italy), this study focused on Matthiola incana, an edible species used in the traditional medicine of various countries. Herein, the characterization of phenolic and volatile compounds, the antioxidant capacity in vitro (1,1-diphenyl-2-picrylhydrazil (DPPH), reducing power and Fe 2+ chelating activity assays) and the toxicity test (Artemia salina lethality bioassay) of the hydroalcoholic extracts from the aerial parts of M. incana subsp. rupestris from Mt. Pellegrino (Palermo) and Mt. Erice (Trapani), and of M. incana subsp. pulchella are reported. The results are compared with those previously shown for M. incana subsp. incana, to achieve a comprehensive overview of the three subspecies. The HPLC-PDA/ESI-MS and SPME-GC/MS analyses led to the identification of 13 phenolics and 54 volatile compounds. Differences in the qualitative-quantitative profile of these phytochemicals have been highlighted between the M. incana subspecies. The antioxidant tests showed different activity for the extracts, which were found to possess better chelating properties. At last, none of the tested extracts displayed toxicity against brine shrimp larvae. These findings enrich the knowledge on the Matthiola taxa growing wild in Sicily, both from the strictly systematic point of view and for the possible applications as sources bioactive compounds that can be used in the nutraceutical field., (© 2021 The Authors. Chemistry & Biodiversity published by Wiley-VHCA AG, Zurich, Switzerland.)