Actinobacteria are the most biotechnologically valuable prokaryotes and are best known as a source of diverse secondary metabolites. However, there is scarce information on marine actinobacterial diversity in the Caribbean Sea. In this study, the diversity of Actinobacteria was examined through culture-dependent methods in the marine sponges Aplysina fistularis and Aplysina fulva. Sponge specimens were collected by SCUBA diving in the La Parguera Natural Reserve in Lajas, Puerto Rico. A total of 62 isolates of Actinobacteria were obtained from the collected sponge samples and identified through phylogenetic analysis based on the 16S rRNA gene. The isolated Actinobacteria were phylogenetically allocated to 18 genera. A. fulva yielded more Actinobacteria isolates (39) than A. fistularis (24). In terms of genus diversity, a higher variety of Actinobacteria was observed from A. fulva (17 genera) than from A. fistularis (7 genera). Even though a culture-dependent approach can give us valuable insight into the sponge's microbiome and is an important basis for the study of the compounds that are produced by Actinobacteria, this strategy is limited because it does not reveal all the Actinobacteria that may be present in a sponge since a small portion of bacteria are culturable. Moreover, a partial sequence of the 16S rRNA gene often does not contain enough phylogenetic information to provide species-level resolutions. Ecological and phylogenetic studies of marine Actinobacteria will improve our understanding of the actinobacterial diversity associated with different marine ecosystems and could lead to the discovery of useful bioactive compounds.