The gut microbiome of carnivorous fish evolves in tandem with the host to adaptively increase the host fitness in a dynamically-changing environment. In order to identify the beneficial strains in supporting the survival of orange-spotted grouper, Epinephelus coioides in a culture environment, the foregut, midgut and hindgut were analysed in three healthy and two moribund individuals. This study was conducted in the Merbok River, northwest Peninsular Malaysia which is exposed to some degree of pollution. The 16S amplicon sequencing targeting the V3-V4 highly variable region revealed similar community richness among samples but compositional and functional differences between the healthy and moribund fishes in the three gut regions. The findings showed a higher abundance of beneficial microflora, including Halomonas, Curvibacter and Bacillus residing in healthy foreguts, coupled with elevated activities of aromatic compound degradation. In the midguts, more than 70% of microbiota comprised of Photobacterium and Vibrio in most specimens. The higher abundance of Vibrio and Photobacterim in moribund fish hindguts may be correlated with increased chitinolytic activity and altered metabolic pathways. The outcomes of the study provide a fundamental understanding of the gut microbiota structure and suggest the potential use of Halomonas, Curvibacter and Bacillus in the orange-spotted grouper survival in a polluted environment.