We isolated and identified 25 unrecorded bacterial species belonging to the phylum Actinomycetota found in the Republic of Korea. Sequence comparison of 16S rRNA was performed using the NCBI BLAST and EzBioCloud database to identify 25 species, which had a 16S rRNA gene sequence similarity of >98.8% and were allocated as unrecorded species in the Republic of Korea. Among the 25 unrecorded bacterial strains, Streptomyces was the most common with nine species, followed by Leifsonia with two species. Isoptericola, Nocardioides, Dermacoccus, Sinomonas, Patulibacter, Marmoricola, Allobranchiibius, Aldersonia, Actinokineospora, Agromyces, Aeromicrobium, Cellulomonas, and Gordonia with one species each were also found. Twenty-five unrecorded species were excavated in various environments, such as tidal flats, ferns, soil, pine cones, moss, mud, wetlands, and plants. These isolates were characterized on the basis of their phylogenetic, biochemical properties, and morphological data, and species descriptions were provided.