Objective: The main objective of the current investigation is to identify the antagonistic potential of marine sediment actinobacterial population collected from coastal regions of Tamil Nadu and Kerala.Methods: A total of 60 marine sediment samples were collected from 27 locations of coastal Tamil Nadu and Kerala. A total of 85 morphologically distinct actinobacterial strains were isolated and taxonomically characterized. The strains were screened for its antagonistic potential against selected bacteria strains, and the obtained results were statistically analyzed. Further, the strains excelled in producing antimicrobial substances were subjected to electron microscopic and chemotaxonomic studies to get more taxonomic details.Results: The isolates were identified as different species of Streptomyces, Micromonospora, Saccharopolyspora, Actinopolyspora, Nocardia, Nocardioides, Kitasatospora, and the rest of them were categorized as “unidentified.” All the isolates were subjected to antimicrobial assay through cross-streak and well diffusion methods that endorsed 34 (40%) of them to be active against clinical and reference bacterial strains such as Staphylococcus aureus MTCC 3160, S. aureus (methicillin-resistant), Bacillus pumilus NCIM 2327, Escherichia coli MTCC 1698, E. coli, Shigella flexneri MTCC 1457, and Proteus vulgaris. The predominance of Streptomyces (18) was ascertained and followed by Saccharopolyspora (7), Micromonospora (5), Nocardia (2), and Kitasatospora (2) with considerable antimicrobial activities. Statistical analysis revealed that the isolates BDK01, BMT01, BPY03, and BRS07 showed significant antimicrobial activities. Phenotypic and chemotaxonomic analysis revealed that all four isolates are belonging under Streptomyces genus.Conclusion: The output of the present investigation revealed that the Streptomyces species are predominant in marine sediments and gain more attention for their antagonistic potential. Therefore, assessment and evaluation of marine microbial diversity were done, which could serve as a potential source for novel antibiotics.