Women's preferences for masculinity in men's faces seem to vary across the menstrual cycle and are assumed to be strongest around ovulation. A number of hormones have been proposed to underlie these subtle cyclic shifts. Furthermore, mating preferences are context-dependent, and stress has been found to alter mate choice, both in animals and humans. Currently, the effects of stress on women's preference for masculinity remain unknown. To examine the hormonal basis and the impact of stress on facial masculinity preference, we tested for within-subject changes in 52 healthy young women who underwent the Trier Social Stress Test (TSST) and the placebo-TSST in randomized order in the late follicular and mid-luteal phases of their menstrual cycle. Menstrual cycle phase and hormone levels were confirmed using estradiol, testosterone, progesterone, and cortisol analyses from saliva. Results show that women were more likely to be attracted to masculine-faced men right before ovulation than in the mid-luteal phase. Estradiol modulated this masculinity preference with high estradiol levels being related to stronger masculinity preference. When stressed however, women experienced a decrease in male facial masculinity preference. In line with these findings, the higher the cortisol increase to stress, the less were masculine faces preferred to more feminine faces. Mate choice is a central component of reproduction. The present results provide information about the effects of stress and hormonal influences on mate preferences in women.