Novel nereistoxin derivatives containing phosphonate were synthesized and characterized via31P,1H and13C NMR and HRMS. The anticholinesterase activity of the synthesized compounds was evaluated on human acetylcholinesterase (AChE) using the in vitro Ellman method. Most of the compounds exhibited good inhibition of acetylcholinesterase. All of these compounds were selected to assess their insecticidal activity (in vivo) againstMythimna separataWalker,Myzus persicaeSulzer andRhopalosiphum padi. Most of the tested compounds displayed potent insecticidal activity against these three species. Compound7fdisplayed good activity against all three insect species, showing LC50values of 136.86 μg/mL forM. separata, 138.37 μg/mL forM. persicaeand 131.64 μg/mL forR. padi. Compound7bhad the highest activity againstM. persicaeandR. padi, with LC50values of 42.93 μg/mL and 58.19 μg/mL, respectively. Docking studies were performed to speculate the possible binding sites of the compounds and explain the reasons for the activity of the compounds. The results showed that the compounds had lower binding energies with AChE than with the acetylcholine receptor (AchR), suggesting that compounds are more easily bound with AChE.