Background: The emergence of multidrug-resistant bacteria because of poor understanding of the issue and the misuse of antibiotics has become global health concern. Therefore, the discovery of novel antibacterial drugs is urgently needed. New antibacterial compounds may be found in the Bacillus species, which are abundant in sand dune ecosystems. Herein, we examined samples from the Parangkusumo coastal sand dunes in Indonesia.Methods: Samples were collected from three areas in the sand dunes (the area closest to the sea, the core area of sand dunes, and the area farthest from the sea). The samples were inoculated on Luria Bertani agar. Morphological and molecular identification was performed on the basis of 16S rRNA. The samples’ antimicrobial activity was evaluated with the disc diffusion method and compared with that of opportunistic pathogenic bacteria.Results: Five species of Bacillus were successfully isolated from the Parangkusumo coastal sand dunes. To our knowledge, this is the first report of the isolation of Bacillus aryabhattai in Indonesia. All samples showed antimicrobial activity against pathogenic bacteria. B. velezensis and B. subtilis showed antibacterial activity against Gram-positive bacteria, whereas B. aryabhattai and B. megaterium showed antibacterial activity against Gram-negative bacteria, and B. spizizenii showed antibacterial activity toward Gram-positive and Gram-negative bacteria.Conclusion: Five Bacillus species were successfully isolated from the Parangkusumo coastal sand dunes, Indonesia, and all samples showed antimicrobial activity toward opportunistic pathogenic bacteria. The crude antimicrobial compounds from B. megaterium, B. aryabhattai, B. subtilis, and B. spizizenii showed the highest growth-inhibition activity against E. coli, P. aeruginosa, B. cereus, and S. aureus, respectively.