In this study, taxonomic classification of intestinal microbiota from healthy Barbour’s seahorses (Hippocampus barbouri) was determined as it plays an important role in host nutrition and immunity. Genomic DNA was extracted from the intestinal mucus samples of eleven Barbour’s seahorses, which were subjected to high-throughput sequencing of bacterial 16 S rRNA genes targeting V3-V4 regions. The results showed that there were 302,891 amplicon sequence reads representing all samples, with 46 total operational taxonomic units (OTU). Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria were the most predominant phyla observed, with 13 classes, 22 orders, 28 families, and 35 genera, respectively. The results also revealed that the most abundant OTUs were affiliated with the genus Shewanella. Findings in this study may shed light on further studies in exploring the potential implications of intestinal microbiota to seahorses in terms of health status, development, growth, and survival. This could also be used as baseline data for preliminary screening of bacterial species as potential probiotics.