Chlorpyrifos (CP) is one of the organophosphate insecticides most used worldwide today. Although the main target organ for CP is the nervous system triggering predominantly neurotoxic effects, it has suggested other mechanisms of action as cytotoxicity and endocrine disruption. The risk posed by the pesticide metabolites on non-target organisms is increasingly recognized by regulatory agencies and natural resource managers. In the present study, cytotoxicity and estrogenic activity of CP, and its principal metabolite 3,5,6-trichloro-2-pyridinol (TCP) have been evaluated by in vitro assays, using two mammalian cell lines (HEK293 and N2a), and a recombinant yeast. Results indicate that TCP is more toxic than CP for the two cell lines assayed, being N2a cells more sensitive to both compounds. Both compounds show a similar estrogenic activity being between 2500 and 3000 times less estrogenic than 17β-estradiol. In order to find new toxicity measurement models, yeasts isolated from marine sediments containing CP residues have been tested against CP and TCP by cell viability assay. Of the 12 yeast strains tested, 6 of them showed certain sensitivity, and a concentration-dependent response to the tested compounds, so they could be considered as future models for toxicity tests, although further investigations and proves are necessary.