Since April 2012, doctor fish in the breeding tank and in the quarantine tank in Hanwha Aquaplanet Yeosu Aquarium have been dying, accompanied by diffuse bleeding around the mouth, in the chin, and at the bottom of the abdomen. In this study, the cause of death would be examined through the bacteriological study of doctor fish and the rearing water quality in the aquarium. The water quality and the bacterial counts of the rearing water in the exhibit tank and in the quarantine tank were analyzed once a week, starting from August to November 2014. Water quality was measured based on the following data: temperature was in the range of 24.5~26.8℃, pH at 6.77~7.94, DO at 6.15~8.61 ppm, ammonia at 0~0.93 ppm, nitrite at 0.009~0.075 ppm, and nitrate at 1.1~40.9 ppm. Studies revealed that the differences in these water quality factors were not related to the death of doctor fish. Bacterial counts in the rearing waters of Garra rufa slightly increased to 103~104 CFU/ml, just before the death of the doctor fish. Twelve strains of bacteria were isolated from the dead fish and rearing waters. The isolates were identified as Aeromonas veronii, Citrobacter freundii, Pseudorhodoferax aquiterrae, Shewanella putrefaciens, and Vibrio anguillarum on the basis of 16S rRNA gene sequences. The most dominant species was C. freundii, which showed medium sensitivity to florfenicol and norfloxacin, and was resistant to amoxacillin, doxycycline, oxytetracycline, tetracycline, and trimethoprim. Ten isolates were confirmed to be pathogenic to the doctor fish. Doctor fish infected with C. freundii and S. putrefaciens showed high mortality in the experimental groups. These results indicate that the variation in bacterial numbers in the rearing water was related to the death of doctor fish. C. freundii and S. putrefaciens were directly implicated in causing the death of doctor fish in the aquarium.