The detrimental impacts of plastic nanoparticles (PNPs) are a worldwide concern, although knowledge is still limited, in particular for soil mesofauna. This study investigates the biochemical impact of 44 nm polystyrene PNPs on three soil models- (Oligochaeta), (Collembola) and (Isopoda). Exposure durations of 3, 7 and 14 days (d) were implemented at two concentrations (1.5 and 300 mg kg PNPs). Results revealed PNPs impact on the activities of the glutathione-dependent antioxidative enzyme, glutathione S-transferase (GST) and on the neurotransmitter acetylcholinesterase (AChE) for all three species. Catalase (CAT) played a minor role, primarily evident in at 300 mg kg PNPs (CAT and GST response after 14 d), with no lipid peroxidation (LPO) increase. Even with the antioxidant defence, was the most sensitive species for lipid oxidative damage (LPO levels increased after 7 d exposure to 300 mg kg PNPs). Significant AChE inhibitions were measured already after 3 d to both PNP concentrations in and , respectively. Significant AChE inhibitions were also found in but later (7 d). Overall, the toxicity mechanisms of PNPs involved antioxidant imbalance, being (mostly) the glutathione-associated metabolism part of that defence system. Neurotoxicity, linked to AChE activities, was evident across all species. Sensitivity to PNPs varied: > ≅ This pioneering study on PNPs toxicity in soil invertebrates underscores its environmental relevance, shedding light on altered biochemical responses, that may compromise ecological roles and soil ecosystem fitness.