Bituminaria bituminosa L. is known for producing several compounds with considerable pharmaceutical interest, such as phenylpropanoids, furanocoumarins and pterocarpans. In vitro cultures of seedlings, shoots, and callus have been produced to obtain plant materials useful for the production of these metabolites. The secondary metabolite profile was evaluated by HPLC-DAD. The extracts of all the in vitro material contained the flavonoid daidzein, while plicatin B, erybraedin C and bitucarpin A were found only in the extracts of the in vitro shoots and in wild shoots. The furanocoumarins angelicin and psoralen were found in in vivo and in vitro plants, but in the callus were not detectable. The extracts were also tested for cytotoxic activity in HeLa cell culture; the highest level of cytotoxicity was found in in vitro shoot extracts.