According to the present knowledge, this is the first report on establishing transformed root cultures of Leonotis nepetifolia after Rhizobium rhizogenes-mediated transformation. The preliminary phytochemical analysis showed differences in the content of phenols and flavonoids in transformed and nontransformed roots. The dominant compounds in the analyzed extracts were (+)-catechin (5464 and 6808 µg/g DW), p-coumaric acid (2549 and 4907 µg/g DW), m-coumaric acid (1508 and 2048 µg/g DW) and rosmarinic acid (1844 and 2643 µg/g DW) for nontransformed (LNNR) and transformed (LNTR4) roots, respectively. Initial biological studies carried out on LNNR, and LNTR4 extracts showed a cytotoxic effect on the A549 lung, HCC1937 breast and leukemia NALM-6 cell lines, antioxidants, as well as repair and protection against DNA damage induced by H2O2 in HUVEC cells. Due to the stronger effect of the LNTR4 root extract, which can be a relatively efficient and cheap source of bioactive secondary metabolites, further biological analyses are needed to discover in detail their potentially valuable biological properties