Estrogen receptor (ER) is a major therapeutic target in the treatment of estrogen-related diseases, such as breast cancer. There is a need to develop potent ER ligands capable of selective targeting of cancer cells without affecting normal cells. Blocking ERα action by antagonists and inhibition of steroidogenic enzymes is standard therapy in the treatment of breast cancer for many years. On the other hand, ERβ isoform usually has anti-proliferative and tumor-suppressive functions, so targeting ERβ with specific agonists represents new promising approach not only in breast cancer therapy, but also prostate. Beside anticancer activity of ERβ agonists, their application is considered in the treatment of depression, anxiety and inflammation. In order to obtain potent antiproliferative agents, triazole ring is often incorporated as pharmacophore into the steroid skeleton. This study provides evaluation of binding affinity of novel N(2)-substituted D-condensed steroidal triazoles for ligand-binding domains (LBDs) of ERβ and androgen receptor using yeast-based fluorescent assay. LBD of steroid receptor was expressed in-frame with yellow fluorescent protein (YFP) in Saccharomyces cerevisiae. Upon ligand-binding induced dimerization, fluorescence resonance energy transfer (FRET) between YFP molecules was analyzed by fluorescence spectroscopy and microscopy. We have identified new selective ERβ ligands without androgenic properties, but further experiments are required to determine whether their mechanism of action is agonistic or antagonistic. Having in mind the broad therapeutic potential of specific ERβ ligands, our findings indicate that steroid derivatives containing triazole are promising bioactive compounds in the field of anticancer agents.