Abstract Poultry litter is widely applied as a fertilizer even though it is one of the main antibiotic sources for agricultural soils. Long-term sublethal effects (56 days) on the antioxidant system of Eisenia andrei earthworms following exposure to fluoroquinolone-contaminated poultry litter (enrofloxacin + ciprofloxacin) at 5.0, 10 and 20 g kg-1 were evaluated. The following biomarkers were assessed: superoxide dismutase (SOD), catalase (CAT), glutathione-S-trasferase (GST), reduced glutathione (GSH) and a lipid peroxidation (LPO) proxy. Significant CAT and SOD increases and a moderate positive correlation (ρ = 0.67, p < 0.05) between these enzymes was observed. Glutathione-S-trasferase levels increased significantly at 10 g kg-1, while GSH exhibited a dose-dependent response at 5.0 mg kg-1 (4 - 106%), 10 mg kg-1 (28 - 330 %) and 20 mg kg-1 (45 - 472%). LPO levels exhibited a decreasing trend with increasing poultry litter concentrations of 8 - 170% (5.0 g kg-1), 7 - 104% (10 mg kg-1) and 3 - 6% (20 mg kg-1). A Principal Component Analysis (PCA) highlighted increased SOD and CAT activities, possibly due to increases in reactive oxygen species (ROS) concentrations. Biological Health Status assessments based on the Biomarker Response Index indicate major alterations in the first month of exposure, changing to moderate in the second month. These findings indicate an antioxidant system attenuation trend. It is possible, however, that successive poultry litter applications may reduce the long-term recovery capacity of the evaluated biomarkers.