The activation of biochar (carbonaceous material generated by pyrolysis of biomass) with extracellular enzymes generated from soil biological processes was studied via a 2-month microcosm using earthworms. The isolation of biochar particles (post-incubated biochar) from earthworm-treated soils allowed to confirm an enrichment of biochar with extracellular enzymes associated to biogeochemical (alkaline phosphatase, β-glucosidase and arylsulfatase) and bioremediation pathways (carboxylesterase). The hydrolytic activity of this biochar incubated with earthworms was up to 8 times higher compared with that of control biochar (incubated in earthworm-free soils). More interestingly, the enzyme carboxylesterase was stabilized onto biochar surface as demonstrated by its unchanged hydrolytic activity after multiple water rinses of the post-incubated biochar, and the remaining activity following heat stress. Moreover, this biochar-bound carboxylesterase activity was sensitive to inhibition by organophosphorus (23–100% of control activity) and methyl carbamate pesticides (37–57%), demonstrating that bioactivated biochar with carboxylesterases may inactivate these agrochemicals by providing stable ligands of enzymatic nature.