Objectives The hormonal treatment for endometriosis frequently fails to completely eradicate endometriotic implants. A new therapeutic treatment is needed. This study investigates the in‐vitro effect of Copaifera langsdorffii oil‐resin on human eutopic and ectopic endometrium stromal cell cultures (EuESCs and EctESCs). Methods A nanocomposite system containing the copaiba oil‐resin (NanoCOR) was developed and acute toxicity test was performed. Endometrial stromal cells (ESCs) from non‐endometriotics controls (CESCs), EuESCs and EctESCs were isolated and treated with different concentrations of NanoCOR, at different time intervals to evaluate its effect on cell morphology, proliferation, viability, necrosis and apoptosis induction. Key findings When treated with 50 μg/ml of NanoCOR, the morphology of EctESCs changed, as the actin microfilaments were disorganized, disassembled or disrupted. Moreover, at 24 h of treatment with NanoCOR, the EctESCs viability was inhibited, and a significant number of these cells underwent apoptosis. In EuESCs, these effects were observed only at 48 h. Finally, the treatment of EctESCs with NanoCOR increased the lactate dehydrogenase release into the extracellular medium more than in EuESCs. Conclusions Our data indicate that NanoCOR has a greater impact on the behaviour of human endometriotic stromal cells than on the eutopic endometrium stromal cells, supporting the idea that NanoCOR should be further investigated as a novel and valuable alternative to treat endometriosis.