Some plant species belonging to Trifolium L. genus are a source of isoflavones considered to exert phytoestrogenic activities. The aim of the present study was to examine the effects of standardized extract obtained from aerial parts of Trifolium medium L., in comparison with the extract of Trifolium pratense L., on the development of estrogen deficiency-induced osteoporosis in rats. Both Trifolium extracts, at doses corresponding to 10 and 20 mg/kg of isoflavone aglycones daily, or estradiol (0.2 mg/kg daily), were administered orally to ovariectomized (OVX) rats for 4 weeks. Serum bone turnover markers, bone mass, mineralization, and mechanical properties were studied. In OVX control rats, mechanical properties of the tibial metaphysis and femoral neck were strongly worsened in comparison with sham-operated control rats, and those of femoral diaphysis were unaffected. Estradiol counteracted the worsening of the tibial strength and increases in bone turnover markers. Both extracts significantly increased the strength of the femoral diaphysis and calcium and phosphorus content in the bone mineral, but only T. pratense extract increased the strength of the tibial metaphysis. In conclusion, effects of both Trifolium extracts differed from those of estradiol. It is possible that other than isoflavone extract constituents contributed to their skeletal effects.