 The interest in the ecological relevance of risk assessments and, thus, in in situ bioassays has been increasing in the last years. The present study developed a time- and cost-effective in situ bioassay, aiming at obtaining, in a short period of time and with a minimum of resources, a set of ecologically relevant toxicological information in a site-specific approach. Poecilia reticulata and Gambusia holbrooki were chosen as test species. Post-exposure feeding inhibition and the biomarkers acetylcholinesterase, lactate dehydrogenase and glutathione S-transferases were the endpoints tested. The battery of biomarkers as a whole was sensitive to the in situ exposure in an acid mine drainage impacted effluent, although responses varied between test species. Post-exposure feeding inhibition was the most sensitive endpoint, and its association with biomarker responses was discussed. The linkage between individual responses, such as feeding, and biomarkers suggested that, at least in this case, biomarkers can be relevant at higher levels of biological organization. Altogether, the proposed short-term in situ bioassay seems to be a promising tool, since it represents a reasonable compromise between sensitivity, time/cost-effectiveness and ecological relevance.