The bacterial communities associated with the gastrointestinal tract are primarily involved in digestion, physiology and immune response against pathogenic bacteria for the overall development and health of the host. Hilsa shad (Tenualosa ilisha), a tropical anadromous fish, found predominantly in Bangladesh and India, has so far been poorly investigated for its gut bacterial communities. In this study, both culture-based and metagenomic approaches were used to detect intestinal isolates of hilsa, captured from both freshwater and seawater to investigate the community structure of intestinal microbiota. Culture-dependent approach allowed to isolate a total of 23 distinct bacterial species comprising sixteen Gram-negative, and seven Gram-positive isolates, where Proteobacteria and Firmicutes were identified as the two most dominant phyla. While metagenomic approach explored a wide range of important gastrointestinal bacteria, primarily dominated by Proteobacteria, Firmicutes and Bacteroidetes, with Proteobacteria and Firmicutes, being the most abundant in freshwater and seawater samples, respectively. A combination of these approaches provided the differential gastrointestinal-associated bacterial diversity in freshwater and seawater hilsa with the prediction of overall functional potential.