Cancer is one of the top causes of death in humans. The currently available treatments, that depend on the type of cancer, may include cytostatic drugs. Methotrexate (MTX) and 5-Fluorouracil (5-FU) are cytostatic drugs that have been used for chemotherapeutic treatment. Due to their nature, their presence in water systems, even in low concentrations, is of particular interest due to the potential risks to non target organisms and humans, through consumption of contaminated items like drinking water. However, knowledge of its impact to aquatic organisms is limited. Thus, this study aimed to evaluate the effects of MTX (0.01 up to 1000 µg.L^-1), 5-FU (0.01 up to 1000 µg.L^-1) and its mixture (10 up to 1000 µg.L^-1) on zebrafish (Danio rerio) focusing on different endpoints, such as fish development (Fish Embryo Toxicity (FET) assay and heartbeat rate), behaviour (light/dark stimulus) and biochemical endpoints associated with neurotransmission (AChE), antioxidant defences (CAT and GST) and metabolism pathways (LDH). Overall, zebrafish embryos demonstrated sensitivity in all tests. Exposure to MTX promoted increased heartbeat rate and swimming behaviour (between 0.01-100 µg.L^-1), however a decrease was observed at the highest concentration (1000 µg.L^-1). Our findings demonstrate that MTX and 5-FU use similar pathways both in humans and zebrafish, once MTX induced disruption in the heartbeat and behaviour and 5-FU induced disruption in behaviour. The mixture of compounds demonstrated an increase impact in the tested parameters.