The present study investigates the toxicity of the herbicide tribenuron methyl (TBM) as an anthropogenic agent and ammonia as an abiotic factor on Daphnia magna at environmentally relevant concentrations. These stressors may coexist in surface waters in agricultural regions. To achieve this objective, D. magna were exposed to TBM at a nominal concentration of 0.81 μg/L in association with a low ammonia (LA) concentration of 0.65 mg/L and a high ammonia (HA) concentration of 1.61 mg/L in acute toxicity tests of 96-h duration and chronic toxicity tests of 21-day duration. The D. magna also were exposed to TBM, HA, and LA singly. The D. magna were analysed for various biomarkers of sublethal toxicity. Glutathione peroxidase (GPx), glutathione S-transferase (GST), cholinesterase (ChE) enzyme activities, and levels of thiobarbituric acid reactive substances (TBARS) and total protein were determined spectrophotometrically. Mitochondrial membrane potential (MMP) was analysed by microscopy with fluorescence staining. Cytochrome c and 5' AMP-activated protein kinase (AMPK) were analysed by Western blotting. Morphometric properties were examined microscopically. This is the first study in which AMPK, an indicator of intracellular energy, was measured in D. magna. GST and ChE enzyme activities and TBARS and total protein levels did not change during acute exposures (i.e., 96 h) in all treatments. GPx activity increased in D. magna from the HA + TBM treatment compared with single-exposure groups. The level of cytochrome c protein was elevated in D. magna from the LA and LA + TBM treatments. AMPK protein levels increased in all treatments with daphnids, except in the LA group. MMP was depolarised in D. magna from all treatments, whereas the most notable change was observed in HA + TBM mixture group in chronic exposures. The results show that GST and ChE may not be sensitive biomarkers for evaluating the sublethal toxic effects to D. magna exposed to environmentally relevant concentrations of ammonia and TBM. Acute and chronic exposure to ammonia and TBM probably caused an energetic crisis in D. magna. Therefore, AMPK and MMP are promising biomarkers for these toxicants.