The reprocessing of spent nuclear fuel (SNF) is crucial for sustainable nuclear energy development. Efficient extraction and separation of actinides from SNF have garnered significant attention in nuclear research. Mixed extractants, such as Et-Tol-DAPhen, which combine soft and hard donors, exhibit selectivity in extracting transuranium elements. In this study, we investigated three N, O-hybrid donor ligands: L1 (dimethyl pyridine-2,6-dicarboxylate), L2 (dimethyl 1,10-phenanthroline −2,9-dicarboxylate), and L3 (dimethyl [2,2′,6′,2″-terpyridine] −6,6″-dicarboxylate). We systematically compared their bonding ability and separation efficiency with Np(III), Pu(III), Am(III) and Cm(III) using the DFT method. Molecular orbital and electrostatic potential analyses revealed that L2 exhibited strong coordination ability than L1 and L3. Bonding analysis indicated decreased covalency of metal–ligand bonds from Np to Cm, attributed to diminishing involvement of 5f orbitals. Thermodynamic analysis demonstrated that these ligands exhibited good extraction effects and successfully separated Cm from Am and Pu. These findings provide valuable guidance for developing efficient extractants.