AbstractWidespread distribution of a highly pathogenic Edwardsiella ictaluri strain in farmed tilapia in northern Vietnam has recently been reported. The subsequent investigation noticed a disease outbreak occurred at five nearby tilapia farms with floating cages, in which the clinical signs of both edwardsiellosis and columnaris diseases were observed on the same infected fish and caused 65% to 85% fish mortality. Naturally diseased fish (n = 109) were sampled from the five infected farms for bacterial identification and conducting challenge tests. The two bacteria Edwardsiella ictaluri and Flavobacterium oreochromis were identified by a combination of biochemical tests, PCR and 16SrRNA sequencing methods. Experimental challenge tests on Nile tilapia resulted in the median lethal dose (LD50) of E. ictaluri and F. oreochromis at 70 CFU/fish by intraperitoneal (i.p.) injection and 3.6 × 106 CFU/mL by immersion, respectively. The experimentally co‐infected challenged fish exposed to LD50 doses resulted in 83% ± 6% mortality, with the infected fish exhibiting clinical signs of both edwardsiellosis and columnaris diseases, mimicking the naturally diseased fish. This finding suggests that the co‐infection of E. ictaluri and F. oreochromis may interact in a synergistic manner, to enhance the overall severity of the infection and elevates the need for efficient methods to control both pathogens.