In recent years, the use of biochemical markers, especially in the assessment of toxic effects and modes of action, under controlled laboratory conditions has increased. However, transposing their use to in situ monitoring or risk assessment evaluations has encountered barriers, mainly related to the difficulty in interpreting the meaning of biochemical variation. In this work, we aimed at understanding if biochemical marker activities (cholinesterase, glutathione S-transferase and lactate dehydrogenase) can be used to monitor the health status of natural populations of fish (Gambusia affinis) and daphnids (Daphnia magna). For that, two ponds with different water properties were chosen as study sites, and organisms collected at four sampling periods along the year. The pattern of biochemical marker responses was not the same in the two species, showing higher integrated biochemical marker response values in the winter for G. affinis and in the autumn for D. magna, suggesting specificities that must be taken into account in biomonitoring programmes by including representative species of several trophic levels. In the case of G. affinis, the differences in key physicochemical parameters between the two ponds (especially dissolved oxygen levels) did not seem to affect biochemical marker levels as if organisms were already perfectly adapted to their environment. In general, seasonal variation of water quality seems to have an important role on biochemical marker responses. Several parameters above Environmental Quality Standards were identified such as dissolved oxygen (DO), ammonia, nitrites, sulphides and metals, but eventual responses to these stressors could not be discriminated from natural variation except for particular cases.