Poultry litter is one of the main sources of fluoroquinolones (FQs) in agricultural soils. In this study, our main goal was to investigate FQ-contaminated poultry litter effects on Eisenia andrei earthworms. To achieve this, acute and chronic tests covered several endpoints, such as avoidance, biomass, lethality, reproduction and changes to immune cells. FQs (enrofloxacin and ciprofloxacin) were determined in a poultry litter sample through high performance liquid chromatography with a fluorescence detector. The avoidance test indicates that poultry litter strongly repels earthworms, even at the lowest concentration (50 g kg− 1 ). In the acute test, the lethal concentration of poultry litter to 50% of the earthworms (LC50), was estimated at 28.5 g kg− 1 and a significant biomass loss (p < 0.05) occurred at 40 g kg− 1 . In the chronic test, a significant reproduction effect was observed at 20 g kg− 1 . Cell typing, density and feasibility indicated significant effects ranging from 5 to 20 g kg− 1 . A high risk quotient was estimated based on recommended poultry litter applications in field studies. Although FQ contamination in poultry litter and soils has been widely reported in previous studies, this is, to the best of our knowledge, the first toxicological assessment concerning earthworms exposed to FQ-contaminated poultry litter.