Many Gram-negative bacteria employ N-acylhomoserine lactones (AHLs) as quorum sensing (QS) signal molecules to regulate virulence expression in a density-dependent manner. Quorum quenching (QQ) via enzymatic inactivation of AHLs is a promising strategy to reduce bacterial infections and drug resistance. Herein, a thermostable AHL lactonase (AidB), which could degrade different AHLs, with or without a substitution of carbonyl or hydroxyl at the C-3 position, was identified from the soil bacterium Bosea sp. F3-2. Ultra-high performance liquid chromatography analysis demonstrated that AidB is an AHL lactonase that hydrolyzes the ester bond of the homoserine lactone ring. AidB was thermostable in the range 30 to 80 °C and showed maximum activity after pre-incubating at 60 °C for 30 min. The optimum temperature of AidB was 60 °C, and the enzyme could be stably stored in ddH₂O at 4 °C or room temperature. AidB homologs were only found in Rhizobiales and Rhodospirillales of the α-Proteobacteria AidB_At from Agrobacterium and AidB_Rm from Rhizobium (amino acid identities of 50.6% and 52.8% to AidB, respectively) also showed thermostable AHL degradation activity. When introduced into bacteria, plasmid-expressed AidB attenuated pyocyanin production by Pseudomonas aeruginosa PAO1 and the pathogenicity of Pectobacterium carotovorum subsp. carotovorum Z3-3, suggesting that AidB is a potential therapeutic agent by degrading AHLs.IMPORTANCE Quorum-sensing system using AHLs as the signal in many bacterial pathogens is a critical virulence regulator and an attractive target for anti-infective drugs. In this work, we identified a novel AHL lactonase AidB from a soil bacterial strain Bosea sp. F3-2. The expression of aidB reduced the production of AHL signals and QS-dependent virulence factors in Pseudomonas aeruginosa and Pectobacterium carotovorum The homologs of AidB with AHL degrading activities were only found in several genera belong to α-Proteobacteria Remarkably, AidB is a thermostable enzyme retained its catalytic activity after treatment at 80°C for 30 min and exhibits reliable storage stability at both 4 °C and room temperature. These properties might make it more suitable for practical application.