Microorganisms are crucial in the bioremediation of organophosphorus pesticides. However, most functional microorganisms (> 99%) are yet to be cultivated. This study applied two cultivation-independent approaches, DNA-SIP and magnetic-nanoparticle mediated isolation (MMI), to identify the functional microorganisms in degrading dimethoate in agricultural soils. MMI identified five dimethoate degraders: Pseudomonas, Bacillus, Ramlibacter, Arthrobacter, and Rhodococcus, whereas DNA-SIP identified three dimethoate degraders: Ramlibacter, Arthrobacter, and Rhodococcus. Also, MMI showed higher resolution than DNA-SIP in identifying functional microorganisms. Two organic phosphohydrolase (OPH) genes: ophC2 and ophB, were involved in dimethoate metabolism, as revealed by DNA-SIP and MMI. The degradation products of dimethoate include omethoate, O,O,S-trimethyl thiophosphorothioate, N-methyl-2-sulfanylacetamide, O,O-diethyl S-hydrogen phosphorodithioate, O,O,O-trimethyl thiophosphate, O,O,S-trimethyl thiophosphorodithioate, and O,O,O-trimethyl phosphoric. This study emphasizes the feasibility of using SIP and MMI to explore the functional dimethoate degraders, expanding our knowledge of microbial resources with cultivation-independent approaches.