Penthiopyrad is a chiral fungicide widely used to control a lot of pathogenic fungi on crops. This study presents six dominant strains of bacteria isolated from the soil with penthiopyrad application history. By employing morphological and genomic methods, the strains were identified as Pseudomonas sp. (PG1), Paenarthrobacter nicotinovorans (PG2), Acinetobacter sp. (PQ1), Serratia marcescens (PQ2), Pandoraea sp. (PQ3), and Burkholderia sp. (PQ4). In the mineral salt medium, the strains exhibit the capacity to degrade penthiopyrad with degradation range of 60%-75% and a significant reduction of the half-life of penthiopyrad stereoisomers (> 17 days). In the soil treatments, the presence of six strains can enhance the degradation of penthiopyrad with the half-life decreasing by 2.6-30.4 days. Four potential metabolites and three possible pathways (hydroxylation, hydrolysis, and carboxylation) were proposed in the treated samples. This study holds promise in providing valuable insights for the development of biodegradation technology, which aims to address soil and environmental contamination resulting from penthiopyrad.