Antibiotic residues from animal manure cause soil pollution and can pose a threat to soil animals. In this study, the toxicological effects of fluoroquinolone antibiotics on Enchytraeus crypticus, including defence response, gut microbiome, and antibiotic resistance genes (ARGs), were studied. The cytochrome P450 enzyme activity and reactive oxygen species levels increased, activating the defense response. The superoxide dismutase and glutathione S-transferase activity, and the expression of immune defense molecules such as coelomic cytolytic factor, lysozyme, bactericidal protein fetidins and lysenin changed. Furthermore, the diversity of the gut microbiome decreased, and the relative abundance of Bacteroidetes decreased significantly at the phylum level but increased in pathogenic and antibiotic-secreting bacteria (Rhodococcus and Streptomyces) at the genus level. However, the soil microbiome was not significantly different from that of the control group. The relative abundance of ARGs in the gut and soil microbiome significantly increased with enrofloxacin concentration, and the fluoroquinolone ARGs were significantly increased in both the soil (20.85-fold, p < 0.001) and gut (11.72-fold, p < 0.001) microbiomes. Subtypes of ARGs showed a positive correlation with Rhodococcus, which might increase the risk of disease transmission and the probability of drug-resistant pathogens. Furthermore, mobile genetic elements significantly promote the spread of ARGs.