While disobeying environmental regulations of Pakistan, several Pb-acid batteries recycling and repairing units discharge their effluents into water canals that irrigate arable fields. Resultantly, serious ecological risks, as well as human health hazards through consumption of edible crops grown on such Pb-polluted soils have been reported. In this experiment, we observed associative effects of amending a soil polluted from Pb-acid batteries effluents (SPB) with arbuscular mycorrhizal fungi (AMF) and lignin-derived biochar (LBC) on barley grain safety to human health. The SPB was treated with AMF inoculum (a consortium of four AMF species), lignin (LN), and LBC, as sole treatments and AMF inoculum with LN and LBC. Barley parameters involving Pb distribution in grain and other parts, grain biochemistry, and nutrition were assessed. Likewise, Pb bioavailability in SPB, AMF root colonization, soil enzymes, microbial biomass carbon (MBC), and AMF produced total glomalin related soil protein (TGSP) were also scoped. Additionally, human renal cells (HEK 293) cytotoxicity test was performed by opting barley grain-related Pb concentrations. Results show that LBC + AMF significantly reduced grain Pb concentrations below the critical limit 4.67 mg kg−1 (WHO/FAO standard), AMF colonization, MBC, soil enzymology, and TGSP, compared to control. Likewise, rest barley parameters were also improved in this treatment. Contrary to other treatments, grain produced on LBC + AMF did not result in (a) cell apoptosis, (b) cell distortion and (c) cohesion loss. Immobilization of Pb in SPB was due to the dilution effect of Pb adsorption on LBC, AMF mycelium and TGSP which resulted in a significant drop of grain Pb concentrations below the critical limit and ultimately no harm to HEK 293 cells. Our findings endorse that grain produced at LBC + AMF treatment are safer for human consumption and will not pose health risks. The LBC + AMF application can remediate SPB for safer cereal production. Display omitted •Soil Pb pollution from acid batteries effluent (SPB) poses health risks via food.•Arbuscular mycorrhizal fungi (AMF) + lignin biochar (LBC) reduced bioavailable Pb.•Barley grain Pb concentrations were below critical limit of WHO/FAO with LBC + AMF.•Highest HEK 293 cells' viability exposed to grain Pb concentrations was at LBC + AMF.•LBC + AMF can produce barley on SPB that will have no harmful effect on human health.