Phoma black stem and leaf spot disease of annual Medicago spp., caused by Phoma medicaginis, not only can devastate forage and seed yield, but also reduces herbage quality by inducing production of phytoestrogens, particularly coumestrol and 4'-O-methylcoumestrol, that can reduce ovulation rates of animals grazing infected forage. We determined the consequent phytoestrogen levels on three different annual Medicago species/cultivars (M. truncatula cv. Cyprus, M. polymorpha var. brevispina cv. Serena and M. murex cv. Zodiac), following inoculation with 35 isolates of P. medicaginis. Across the isolate x cultivar combinations, leaf disease incidence (%LDI), petiole disease incidence (%PDI), leaf disease severity (%LDS), petiole disease severity (%PDS), and leaf yellowing severity (%LYS) ranged up to 100, 89.4, 100, 58.1 and 61.2%, respectively. Cultivars Cyprus and Serena were most susceptible and cv. Zodiac the most resistant to P. medicaginis. Isolates WAC3653, WAC3658 and WAC4252 produced the most severe disease. Levels of phytoestrogens in stems ranged from 25 to 1995 mg/kg for coumestrol and from 0 to 418 mg/kg for 4'-O-methylcoumestrol. There was a significant positive relationship of disease incidence and severity parameters with both coumestrol and 4'-O-methylcoumestrol contents, as noted across individual cultivars, and as across the three cultivars overall where r=0.39 and 0.37 for coumestrol and 4'-O-methylcoumestrol, respectively (P<0.05). Although, cv. Serena was most susceptible to P. medicaginis and produced the highest levels of phytoestrogens in the presence of P. medicaginis, cv. Zodiac contained the highest levels of phytoestrogens in comparison with other cultivars in the absence of P. medicaginis. There was a 15-fold increase in coumestrol in cv. Serena but only a 7-fold increase in cv. Zodiac from infection of P. medicaginis. The study highlights that the intrinsic ability of a particular cultivar to produce phytoestrogens in the absence of the pathogen, and its comparative ability to produce phytoestrogens in the presence of the P. medicaginis, are both important and highly relevant to developing new annual Medicago spp. cultivars that offer improved disease resistance and better animal reproductive outcomes.