Infectious spleen and kidney necrosis virus (ISKNV) and nervous necrosis virus (NNV) are two common and important causative agents in marine-cultured fish. However, high viral loads of both ISKNV and NNV in the same clinical case is unusual. In this study, a mass mortality event of Asian seabass Lates calcarifer juveniles occurred in Zhuhai, the main Asian seabass cultured area in mainland China. The fish samples were pooled for pathogen identification and both high viral loads of ISKNV and NNV were detected by real-time microfluidic quantitative PCR and conventional PCR. Immunohistochemistry and immunofluorescence showed that strong ISKNV signals were detected in spleen and liver, while strong NNV signals were detected in brain and eye. The tissue homogenates were inoculated into MFF-1 cell and SSN-1 cell, respectively. After several viral passages, both individual ISKNV and NNV were purely isolated from each other, and designated as ASB-ISKNV-23 and ASB-NNV-23, respectively. The whole genome sequences of ASB-ISKNV-23 and ASB-NNV-23 were determined and annotated. The result showed that ASB-ISKNV-23 and ASB-NNV-23 are composed of 112,236 bp and 4441 bp, respectively. Phylogeny analysis showed that ASB-ISKNV-23 belongs to ISKNV-II sub-genotype and ASB-NNV-23 belongs to RGNNV genotype. Collectedly, coinfection of ISKNV-II and RGNNV were firstly documented in mass mortality of Asian seabass in mainland China. Importantly, both individual ISKNV-II and RGNNV were purely isolated using two different permissive cell lines. Our study provides useful information for better understanding the complex pathogenesis regarding the coinfection with ISKNV and NNV in farmed fish.