The investigation presented a novel finding regarding mitigating stress induced by chlorpyrifos in Eisenia fetida by incorporating biochar derived from rice straw and fly ash as soil amendments. It was observed that phenolic compounds exhibit solubility in methanol, and the methanolic fraction exhibited notable inhibitory effects on lipid peroxidation and displayed antioxidant properties. The defence mechanism of E. fetida, comprising catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), and various other enzymes, remained effective in neutralizing stressors without disruption when the earthworm was subjected to diverse chemical agents or stressful conditions. These enzymes served as indicative markers of toxicity induced by pesticide exposure, even at sublethal concentrations. The scavenging of free radicals by these enzymes ultimately safeguarded the organism. Fly ash and biochar emerged as two organic alternatives capable of alleviating stress by providing a protective mechanism. In this context, the study examined the impact of biochar and fly ash amendments on earthworm biomarkers. The mortality rate at the median lethal concentration of chlorpyrifos was reduced to less than 50% through 3% and 5% modifications. In contrast to the non-amendment group exposed to sublethal doses, the amendment group exhibited higher levels of oxidative stress and lower protein content. This observation indicated the presence of stress induced by the accumulation of free radicals, which increased in number with higher doses of chlorpyrifos. Moreover, the study highlighted the interconnected nature of total antioxidant capacity and total phenolic capacity values, with a decrease in these parameters signifying a shift in earthworm biomarkers.Graphical abstract