Several bacteria have been isolated from different tropical ecosystems (tropical rain forest, seawater, and marine sediments) from the island of Puerto Rico. These microorganisms were screened for carbohydrase activity using chromogenic substrates (amylose, pullulan, cellulose, dextran, arabinan, galactomannan, and xylan). The results indicate that 2–9% of cultured organisms show carbohydrase activity. Isolates were identified by partial sequence analysis of the polymerase chain reaction amplified small subunit rRNA gene. GenBank database comparison of the most versatile strains gave a match with Bacillus sp., Staphylococcus sp., Halomonas sp., and a gamma proteobacterium. Growth curves indicate that all strains assayed can grow as well in at least one complex carbohydrate as in glucose. A high β-xylosidase activity was detected in the Bacillus sp. strain crude enzyme preparation and this activity seems to be almost entirely cell associated. Our results show that tropical environments can be potentially good sources of bacteria with novel carbohydrases.