Pollution of river systems in Tunisia by endocrine disrupting compounds (EDCs) is due to anthropogenic impacts through non-treated industrial wastewater and municipal-treated wastewater discharge. The aim of this study was to assess the estrogenic activity of environmental matrices involving raw industrial wastewater (WW), surface water (SW), and treated wastewater (TWW). A modified E-screen assay was used to evaluate estrogenic activity of the samples in MCF-7 breast cancer cells. Western blotting and real-time PCR molecular approaches were applied to investigate the mechanism of EDC-induced MCF-7 breast cancer cell proliferation which interferes with estrogen receptor alpha (ERα) and c-Jun. Our results revealed a dose-dependent estrogenic activity reaching an optimal level at a concentration of 0.01% for industrial WW and SW and 10% for TWW. Monitoring ERα protein and c-Jun gene expression levels after 1, 6, 24, and 48 h of treatment showed a correlation in the overexpression of ERα and c-Jun most of the time. In fact, ERα and c-Jun correlated up-regulation began after 1 h of treatment for industrial WW, TWW, and one SW sample. For the other SW samples, this correlated up-regulation started after 6–24 h of treatment. Results emphasized the estrogen-like effect of our contaminated samples on MCF-7 cells which facilitate a cross-talk between ERα and c-Jun factor, thereby directly regulating the expression of estrogen-induced genes to mediate breast cancer cell growth.