Environmental contamination from landfill leachate has been linked to disturbances in human health. However, only global parameters such as dissolved organic content (DOC), chemical oxygen demand (COD), and biological oxygen demand (BOD) are often used to evaluate wastewater quality. In this study, we determined leachate cytotoxicity and stress response of leachate-treated mammalian cells using in vitro bioassays and other molecular techniques. The modified E-screen assay using human breast cancer MCF7 cells was used to determine the estrogenic potential and/or cytotoxicity of water samples from two solid waste landfills in Tunisia. The cytotoxicity mechanism of the leachate was determined by DNA fragmentation and lactate dehydrogenase assays. The stress response of heat shock protein (HSP) 47-positive Chinese hamster ovary cells treated with leachate was also determined. Proteomics analyses and real-time reverse transcription-polymerase chain reaction (RT-PCR) were used to determine and confirm the enhanced expressions of certain stress-related proteins. Results showed that the leachate samples generally do not have estrogenic activity; instead, they are cytotoxic towards MCF7 cells and the cytotoxicity is by necrosis during the early stages of incubation. Leachate also enhanced the expression of heat shock protein (HSP) 47, and various stress-related proteins such as heterogeneous nuclear ribonucleoprotein (hnRNP) E-1, phosphoglycerate mutase, and nuclear matrix protein (NMP) 200 in MCF7 cells. These can be considered as survival mechanisms against leachate-induced cytotoxicity.