Abstract: 【Objective】 In order to clarify the pathogen of gill rot of Oxyeleotris marmoratus in a farm in Zhuhai City， Guangdong Province in May 2022，and to provide reference for prevention and control of the related diseases of O. marmoratus.【Method】 Bacteria were isolated from the rotten gill，kidneys and surface ulceration of the diseased fish，and the dominant pathogen was purified，and the 16S rRNA gene of the isolate was amplified by PCR，and then was sequenced and analyzed.Furthermore，phylogenetic tree of the 16S rRNA gene was constructed to identify pathogenic bacteria. A representative strain Om2202 was selected for detection of virulence genes and artificial infection test of Danio rerio. In addition，the antibacterial and bactericidal activities of Chinese herbal medicines and disinfectants against the isolate Om2202 were determined，and the pathogenicity and drug susceptibility of strain Om2202 were analyzed.【Result】 The dominant strains isolated from the gill and kidney of the diseased fish were slender curved or straight rod-like，without flagella and fimbriae，with capsules and extracellular secretions. The isolate Om2202 was identified as Flavobacterium columnare based on specific PCR，phylogenetic tree analysis of 16S rRNA gene，and colony morphology analysis. The results of detection of virulence genes showed that the strain Om2202 carried 11 virulence-related genes including gldD， hly，cslA， ompA and chiA. The results of pathogenicity test showed that Om2202 strain had strong pathogenicity to D. rerio，and its LC₅₀ was 7.50×10⁶ CFU/mL. The results of drug susceptibility test showed that strain Om2202 was sensitive to aloe-emodin，emodin and benzalkonium bromide，but not sensitive to Forsythia suspensa essential oil，chlorine dioxide，methionine iodine and calcium peroxide.【Conclusion】 F. columnare is the main pathogenic bacteria causing the gill rot of O. marmoratus，which belongs to the widespread genotype II and carries a large number of virulence-related genes. It has a strong pathogenicity to D. rerio and can be controlled with aloe-emodin，emodin and benzalonium bromide.