Nowadays, there is growing evidence that many compounds that were once considered safe, interfere with the endocrine system and act as endocrine disruptors. Among these compounds are also active substances of drugs that are used on a daily basis for the treatment in human or veterinary medicine. These active substances and their metabolites do not only present a problem for the patients and animals, receiving them, but also for all other living creatures. Due to the increased use of pharmaceuticals, their presence in the environment, where everyone can be exposed to them, is also increasing. Therefore, we decided to check the possible effect of the selected active substances on the endocrine activity. Based on the prediction obtained with the computer program Endocrine Disruptome we decided to evaluate the effect of four anxiolytics – clonazepam, lorazepam, sertraline and mianserin – on the estrogen receptor. The program predicted a high probability of binding or agonistic/antagonistic activity on the estrogen receptor α (sertraline hydrochloride) or estrogen receptor β (clonazepam, lorazepam and mianserine hydrochloride). Clonazepam is used for the treatment of epilepsy, while the other three are used for the treatment of various mental disorders, such as depression and anxiety. In the research, the cell line hERα-HeLa-9903 expressing the functional estrogen receptor α was used to determine the effects of the selected compounds on the ERα. First, a cell viability test was performed for all compounds to determine their maximum non-cytotoxic concentration. Clonazepam and mianserin proved to be non-cytotoxic at the highest tested concentration (100 μM), but for lorazepam and sertraline a corresponding lower concentration had to be found. The first non-cytotoxic concentration of sertraline was 25 μM and of lorazepam was 10 μM. In the second part of the compound testing, a screening luciferase test was performed to determine the agonistic/antagonistic effect of the investigated compounds on ERα. The results of the agonist test showed that none of the investigated compounds was an agonist for ERα, since no significant increase in their luciferase activity was observed. The results of antagonistic testing showed a statistically significant decrease in luciferase activity only at higher concentrations (50-100 μM) of clonazepam. The decrease at these concentrations was higher than the limit for a positive result in the test protocol. Based on that, we can conclude that clonazepam is at higher concentrations an antagonist for ERα, but we should first check the possibility of its action directly on the luciferase enzyme. The results of our experiment did not confirm that the investigated compounds act as estrogen receptor α modulators. Only clonazepam showed a possible ability to modulate the estrogen receptor α.