In this work were studied the phenolic composition of in vitro material (shoots, calli, and roots) of Brassica oleracea var. costata and its antioxidant capacity. Samples were obtained in different culture medium, with distinct supplementations to verify their influence on those parameters. Phenolic determination was achieved by HPLC-DAD. Antioxidant activity was assessed against DPPH. In calli and roots no phenolic compound was identified. In shoots was verified the presence of 36 compounds, which included hydroxycinnamic acids, flavonoids (kaempferol and quercetin derivatives), and hydroxycinnamic acyl glycosides (with a predominance of synapoyl gentiobiosides). MS liquid medium supplemented with 2 mg/L benzylaminopurine (BAP) and 0.1 mg/L naphthaleneacetic acid (NAA) revealed to be the best in vitro condition to produce shoot material with highest phenolic compound contents and stronger antioxidant potential, thus with a possible increase of health benefits.