[Purpose] To evaluate the electrical activity of the pelvic floor muscle in women during the follicular, ovulatory, and luteal phases of the menstrual cycle and its correlation with estradiol and total testosterone levels. [Subjects and Methods] This cross-sectional study involved 30 women with ovulatory menstrual cycles. Total testosterone and estradiol levels were measured and the muscle tone and maximum voluntary contraction of the pelvic floor muscles were evaluated using surface electromyography. [Results] Muscle tone was significantly lower during the follicular (21.1±3.3 μV) and ovulatory (27.1±5.9 μV) phases than the luteal phase (30.4±4.1 μV). The maximum voluntary contraction was not different across phases. The estradiol level on the 7th day of the menstrual cycle showed a strong positive correlation with muscle tone and maximum voluntary contraction, and the testosterone level was positively correlated with muscle tone on the 21st day. [Conclusion] Women have better muscle tone during the luteal phase. The muscle tone and maximum voluntary contraction were strongly correlated with the estradiol level on the 7th day, and the muscle tone was correlated with the testosterone level on the 21st day of the menstrual cycle. These findings suggest that hormonal fluctuations during the menstrual cycle alter pelvic floor muscle activity.