Abstract: Objective This study aimed to conduct isolation, identification, and antimicrobial susceptibility testing of pathogenic bacteria from diseased red-tailed crown fish (Aequidens rivulatus), providing theoretical basis and practical guidance for the prevention and control of diseases in red-tailed crown fish. Methods The strain was identified through morphological observation, physiological and biochemical characteristic analysis, 16S rRNA gene sequencing, and phylogenetic tree construction. Its susceptibility to antimicrobial agents was also determined. Results The results showed that the physiological and biochemical characteristics of the isolated bacterium were consistent with those of Proteus penneri. Meanwhile, molecular identification results and phylogenetic tree analysis indicated that the strain had a close genetic relationship with P. penneri, thus confirming the isolated pathogen as Proteus penneri. In the regression infection test, the isolated strain was found to be pathogenic to red-tailed crown fish. For individual red-tailed crown fish weighing approximately 50 g, the median lethal dose (LD₅₀) was 5.16×10⁵ CFU/fish. Antimicrobial susceptibility testing showed that the strain was sensitive to ciprofloxacin, enrofloxacin, and trimethoprim-sulfamethoxazole, but resistant to penicillin and ceftriaxone. Conclusion In summary, this study confirmed that Proteus penneri is the pathogenic bacterium causing diseases in red-tailed crown fish. On the basis of ensuring the safety of aquatic drug use, enrofloxacin and ciprofloxacin can be used as effective drugs for treating infections in red-tailed crown fish.