Nanoselenium (nano-Se) or melatonin (MT) foliar spray reduces pesticide stress by stimulating plant secondary metabolism and antioxidant capacity. However, the effects of nano-Se and MT biofortification on the interaction between plant secondary metabolic pathways and rhizosphere microbes in mitigating 2,4-D butyrate stress remain unknown. Compared to nano-Se or MT treatment alone, nano-Se and MT combined application increased the antioxidant enzyme activities and decreased the MDA (25.0%) and H 2 O 2 (39.3%) contents with 2,4-D butylate exposure. Importantly, they enhance the soil enzymes (S-FDA by 53.1%), allelochemicals (luteolin by 164.1% and tricin by 147.3%), as well as plant secondary metabolites (JA by 63.3% and 193.3% in leaves and roots) levels. It also improved the beneficial microbial abundance of Comamonadaceae, Sphingomonadaceae, and Rhodobacteraceae in the rhizosphere soil. In conclusion, nano-Se and MT alleviate 2,4-D butylate stress in wheat plants by enabling the interaction between rhizosphere microorganisms, allelopathic substances, and secondary metabolites.