This study investigated the bacterial diversity and Vibrio harveyi distribution associated with diseased fugu (Takifugu rubripes) in northeastern China from January to December in 2014. The main clinical signs included fin ulceration, skin darkness, hepatohemia and intestinal hydrops. Totally, 104 diseased live fish were collected and 70 strains isolated from naturally diseased T. rubripes. Most isolates were obtained in May, September and December. The isolates were identified through 16S rRNA gene sequence analysis and Vibrio spp.-specific PCR amplification, followed by pathogenicity determination. Results showed that the isolates belonged to 10 genera, including Vibrio (72%), Staphylococcus (9%), Pseudomonas (4%), Bacillus (4%), Vagococcus (3%), Shewanella (3%), Planococcus migula (4%), Exiguobacterium (1%), Enterobacter (1%) and Kocuria roseus (1%). Vibrio spp. and Vibrio harveyi were the predominant genus and species, respectively. In addition, challenge tests demonstrated that 13 out of 70 isolates were strongly pathogenic and identified as V. harveyi. This study illustrated that V. harveyi could be considered as main pathogen. These investigation results would provide useful information for disease prevention in T. rubripes culture.