Difenoconazole is a persistent triazole fungicide frequently detected in agricultural environments, where its environmental fate is influenced by soil amendments and soil biota. This study investigates the interactive effects of mealworm frass-derived biochar and the anecic earthworm Lumbricus terrestris on the fate of difenoconazole and soil ecological functions. Using two-dimensional terraria to simulate planar soil profiles, a 30-day mesocosm experiment evaluated eight treatment combinations in a full factorial design. Four amendment treatments (control, 5 mg kg⁻¹ difenoconazole, 2% (w/w) biochar, and a combination of both) were each tested in the presence and absence of Lumbricus terrestris. Soil responses were assessed by measuring nutrient cycling (C, N and P) and a suite of enzyme activities (e.g. β-glucosidase, urease and catalase) within earthworm casts, the drilosphere and bulk soil. Concurrently, the ecotoxicological response of Lumbricus terrestris was evaluated using oxidative stress biomarkers, including GST, GR, GPx, lipid peroxidation (MDA), and mitochondrial oxidative phosphorylation (OXPHOS) in the body wall, gastrointestinal tract and seminal vesicles. This research aims to elucidate how biochar alters fungicide bioavailability and how earthworm bioturbation influences contaminant distribution and microbial interactions. The findings provide critical insights into the potential of biochar–fauna interactions for the remediation of pesticide-contaminated agricultural soils while preserving essential ecosystem services.