Per- and polyfluoroalkyl substances (PFAS) comprise a large class of synthetic chemicals characterized by high thermal and chemical stability, as well as hydrophobic and oleophobic properties. These characteristics have led to their widespread use in numerous industrial and consumer products. Their extensive application has resulted in continuous environmental release, making PFAS ubiquitous contaminants. Due to growing concerns regarding their persistence and toxicity, several legacy PFAS, such as perfluorooctanoic acid (PFOA), have been restricted or phased out and replaced by newer compounds, which are assumed to be less hazardous. However, information on their environmental impacts remains limited. This study evaluated and compared the ecotoxicological effects of a 21-day exposure to PFOA and three emerging molecules (ADV MFS-N2, ADV MFS-N3, and ADV MFS-M3) in Daphnia magna. Organisms were exposed to environmentally relevant concentrations (150, 1500, and 3000 ng/L), selected based on levels detected in the discharge channel of a fluorochemical plant and freshwaters of the receiving river. Effects were assessed at both sub-individual and individual levels by measuring energetic biomarkers (lipids, proteins, carbohydrates, and total caloric content), body size traits, swimming behavior, and survival. PFOA exposure caused slight adverse effects, mainly consisting in a reduction in total body length. In contrast, ADV MFS-N2 induced significant adverse responses across multiple endpoints, indicating a higher toxic potential than the legacy compound. These findings highlight the need for further investigation on the environmental fate and ecotoxicological effects of emerging PFAS, as their widespread occurrence may pose risks to aquatic ecosystems and potentially to human health.