Rhodococcus sp. 3-2 strain has been reported to degrade benzimidazole-based pesticides, such as benomyl and carbendazim. Therefore, this study aimed to optimize culture medium composition and culture conditions to achieve cost-effective and efficient large-scale production of the Rhodococcus sp. 3-2 strain. The study identified that the optimal media composition for mass culture comprised 0.5% glucose, 0.5% yeast extract, 0.15% NaCl, 0.5% K 2 HPO 4 , 0.5% sodium succinate, and 0.1% MgSO 4 . Additionally, a microbial agent was developed using a 1.5-ton fermenter, with skim milk (20%), monosodium glutamate (15%), and vitamin C (2%) as key components. The storage stability of the microbial agent has been confirmed, with advantages of low temperature conservation, which helps to sustain efficacy for at least six months. We also assessed the benomyl degradation activity of the microbial agent within field soil. The results revealed an over 90% degradation rate when the concentration of viable cells exceeded 2.65 × 10 6 CFU/g after a minimum of five weeks had elapsed.