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In a project designed to investigate the specific and infraspecific taxa of <i>Matthiola</i> endemic to Sicily (Italy) as new potential sources of bioactive compounds in this work, the infraspecific taxa of <i>Matthiola fruticulosa</i> were studied, namely, subsp. <i>fruticulosa</i> and subsp. <i>coronopifolia</i>. HPLC–PDA/ESI–MS and SPME–GC/MS analyses of hydroalcoholic extracts obtained from the aerial parts of the two subspecies led to the detection of 51 phenolics and 61 volatile components, highlighting a quite different qualitative–quantitative profile. The antioxidant properties of the extracts were explored through in vitro methods: 1,1-diphenyl-2-picrylhydrazyl (DPPH), reducing power and Fe²⁺ chelating activity assays. The results of the antioxidant tests showed that the extracts possess a different antioxidant ability: particularly, the extract of <i>M. fruticulosa</i> subsp. <i>fruticulosa</i> exhibited higher radical scavenging activity than that of subsp. <i>coronopifolia</i> (IC₅₀ = 1.25 ± 0.02 mg/mL and 2.86 ± 0.05 mg/mL), which in turn displayed better chelating properties (IC₅₀ = 1.49 ± 0.01 mg/mL and 0.63 ± 0.01 mg/mL). Lastly, <i>Artemia salina</i> lethality bioassay was performed for toxicity assessment. The results of the bioassay showed lack of toxicity against brine shrimp larvae for both extracts. The data presented indicate the infraspecific taxa of <i>M. fruticulosa</i> as new and safe sources of antioxidant compounds.