The changes in melatonin concentration in relation to sexual steroid and thyroid hormones in cyclic Egyptian buffaloes were studied. In addition, the beneficial effect of vitamins (AD3E) plus tonophosphan on the subsequent reproductive performance of buffaloes with inactive ovaries as well as the effect upon melatonin in relation to sexual steriod and thyroid hormones were investigated. The present study revealed that, there was a relationship between levels of melatonin and thyroid hormones with sexual steroid hormones during estrous phases. The melatonin was significantly positive correlation with progesterone and significantly negative correlation with estradiol. Moreover, melatonin has a non-significantly negative correlation with thyroid hormones. The percentage of Egyptian buffaloes that exhibited heat after treatment was 73.33% and conception rate was 77.27%. The melatonin was significantly decreased after treatment as well as thyroid hormones arise. In conclusion, there is a role for melatonin in the regulation of estrous cycle. Ovarian inactivity in Egyptian buffaloes seems to be due to poor nutrition which considered a primary factor limiting net reproductive rate, as well as vitamins (AD3E) plus phosphorus (tonophosphan) appears to be the most appropriate treatment.