The possibility of chemical contamination is an important issue to consider when designing a cell therapy strategy. Both bisphenol A (BPA) and 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) are among the most environmentally relevant endocrine disrupting chemicals (EDCs, compounds with a high affinity for adipose tissue) recently studied. Adipose‐derived stem cells (ASCs) are mesenchymal stromal cells (MSCs) obtained from adipose tissue widely used in regenerative medicine to prevent and treat diseases in several tissues and organs. Although the experimental use of tissue‐engineered constructs requires careful analysis for approval and implantation, there has been a recent increase in the number of approved clinical trials for this promising strategy. This study aimed to evaluate cell viability, apoptosis, DNA damage, and the adipogenic or osteogenic differentiation potential of rat adipose‐derived stem cells (rASCs) exposed to previously established non‐cytotoxic doses of BPA and TCDD in vitro. Results demonstrated that 10 μM of BPA and 10 nM of TCDD were able to significantly reduce cell viability, while all exposure levels resulted in DNA damage, although did not increase the apoptosis rate. According to the analysis of adipogenic differentiation, 1 μM of BPA induced the significant formation of oil droplets, suggesting an increased adipocyte differentiation, while both 10 μM of BPA and 10 nM of TCDD decreased adipocyte differentiation. Osteogenic differentiation did not differ among the treatments. As such, BPA and TCDD in the concentrations tested can modify important processes in rASCs such as cell viability, adipogenic differentiation, and DNA damage. Together, these findings prove that EDCs play an important role as contaminants, putatively interfering in cell differentiation and thus impairing the therapeutic use of ASCs.