Although it is known that Palk Bay sediments harbor diverse and novel bacteria with important ecological and environmental functions, a comprehensive view of their molecular diversity is still lacking. In the present study, bacterial diversity in Palk Bay sediments was characterized using the molecular method terminal-restriction fragment length polymorphisms (T-RFLP). The bacterial assemblages detected by T-RFLP analysis revealed that the nearshore sediment harbored high number of bacterial count, whereas the 2.5-m sediment harbored diverse and distinct bacterial composition with fine heterogeneity. The major bacterial groups detected in all the three sediment samples were Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria (including alpha (alpha), gamma (gamma), delta (delta), and epsilon (epsilon)-Proteobacteria), and uncultured bacteria. This is the first study that reveals the presence of Bacteroidetes, delta (delta)- and epsilon (epsilon)-Proteobacteria, and uncultured bacteria in Palk Bay sediments. The hitherto unexplored wide microbial diversity of Palk Bay coastal area was unraveled in the current study through culture-independent approach. These data suggest that the continued use of cultivation-independent techniques will undoubtedly lead to the discovery of additional bacterial diversity and provide a direct means to learn more about the ecophysiology and biotechnological potential of Palk Bay coastal area.