This study employs the DNA barcoding approach to make a molecular taxonomic catalog of reef fishes of Saint Martin's Island (SMI), an ecologically critical area (ECA), and Marine Protected Area (MPA) in Bangladesh. DNA barcoding, along with morphological analysis, confirmed 84 reef‐associated fish species in SMI belonging to 16 orders, 39 families, and 67 genera. A total of 184 sequences were obtained in this study where 151 sequences (534–604 bp) of 81 species were identified from the COI barcode gene and 33 sequences (609 bp) of 19 species from the 16S rRNA gene region which were submitted to the GenBank and Barcode of Life Data System (BOLD). Among these sequences, 70 sequences of the COI gene and 16 sequences of 16S rRNA gene region from 41 species were submitted for the first time into the GenBank from Bangladesh. For molecular characterization analysis, another 37 sequences of 15 reef fish species of SMI were added from previous studies, making a total of 221 DNA sequences which comprised 179 sequences of 96 species for the COI gene and 42 sequences of 26 species for the 16S rRNA gene region. The COI sequences contain 145 haplotypes with 337 polymorphic sites, and the mean genetic distances within species, genera, and families were calculated as 0.34%, 12.26%, and 19.03%, respectively. On the contrary, 16S rRNA sequences comprised 31 haplotypes with 241 polymorphic sites, and the mean genetic divergences within species, genera, and families were 0.94%, 4.72%, and 12.43%, respectively. This study is a significant contribution to the marine biodiversity of Bangladesh which would facilitate the assessment of species diversity for strategizing management action. It is also an important input to the DNA barcode library of reef fishes of the northern Bay of Bengal.