In Egypt, until yet no records are available regarding possible multiple hormonal activities in the aquatic systems and especially in the river Nile. In this paper, in vitro yeast estrogen screen (YES) and yeast androgen screen (YAS) were used to assess (for the first time) the multiple hormonal activities in surface waters and sediments of the river Nile. This study aimed to determine whether river Nile water can cause changes in gonadal histology of Nile tilapia (Oreochromis niloticus niloticus). All water samples exhibited extremely low levels of estrogenicity. Estrogenicity was nearly not detected in any of the sediment samples. Unlike the estrogenicity, significant androgenic activities were recorded in the water and sediment samples along the course of the river Nile. The present study reports for the first time quantification anti-estrogenic and anti-androgenic activities with high levels in both water and sediment of the river Nile. The greatest anti-estrogenic and anti-androgenic activities were observed in samples from downstream river Nile. These results indicated that the anti-estrogenic and anti-androgenic activities along the Nile course were great and the pollution of the sites at downstream was more serious than the upstream sites due to industrial and anthropogenic activities at these sites. Good correlations were observed among some hormonal activities, suggesting coexistence of these contaminants in the environmental matrices. There were no signs of sexual disruption in any of the gonads analyzed from either male or female Nile tilapia, demonstrating that no hormonal activity present along the Nile course was sufficient to induce adverse effects on reproductive development. Further investigation is necessary to identify the compounds responsible for the hormonal activities in the river Nile and to examine effects of very low levels of hormonally active compounds on gonadal histology, as well as in the development of more sensitive biomarkers.