Artificial sweeteners are widely used in food and pharmaceuticals, but their stability and persistence raise concerns about their impact on aquatic life. Although standard toxicity tests do not reveal lethal effects, recent studies suggest a potential neurotoxic mode of action. Using environmentally relevant concentrations, we assessed the effects of sucralose and acesulfame, common sugar substitutes, on Daphnia magna focusing on biochemical (acetylcholinesterase activity; AChE), physiological (heart rate), and behavioural (swimming) endpoints. We found dose-dependent increases in AChE and inhibitory effects on heart rate and behaviour for both substances. Moreover, acesulfame induced a biphasic response in AChE activity, inhibiting it at lower concentrations and stimulating at higher ones. For all endpoints, the EC₅₀ values were lower for acesulfame than for sucralose. Additionally, the relationship between acetylcholinesterase and heart rate differed depending on the substance, suggesting possible differences in the mode of action between sucralose and acesulfame. All observed EC₅₀ values were at μg/l levels, i.e., within the levels reported for wastewater, with adverse effects observed at as low as 0.1 μg acesulfame /l. Our findings emphasise the need to re-evaluate risk assessment thresholds for artificial sweeteners and provide evidence for the neurotoxic effects of artificial sweeteners in the environment, informing international regulatory standards.