The diversity of culture-dependent actinobacteria was investigated and compared between two sponges (Mycale sp. and Styliss sp.) collected from Fujian province and two ascidians (Styela plicata and Molgula manhattensis) collected from Hainan province under the same isolation conditions. Five actinobacteria-selective isolation media and one general bacteria isolation medium were selected to culture actinobacteria from both. The phylogenetic affiliation of the actinobacterial isolates was assessed using a combination of 16S rRNA gene amplification-restriction fragment length polymorphism (RFLP) analysis and 16S rRNA gene sequencing. 198 purified actinobacteria were cultivated (87 from sponges and 111 from ascidians). There were 38 different RFLP patterns and the phylogenetic analysis reveals a high and distinctive diversity of actinobacteria associated with these two sponge and two ascidian species, with six and seven actinomycetes genera, respectively. Apart from Streptomyces genus isolated from both sponge species, four other genera were found only in the Mycale sp. sponge compared to one more genus found in the Styliss sp. sponge. Similarly, apart from the ubiquitous Streptomyces genus, five genera were isolated only from ascidian Styela plicata whereas only one other genus was isolated from ascidian Molgula manhattensis. The 16S rRNA gene sequence analysis showed that two actinobacterial isolates from sponge and eight isolates from ascidians are probably novel strains with similarity lower than 97% compared to the nearest type strains. Except for Arthrobacter genus detected in a sponge sample and two ascidian-specific actinobacterial genera Rhodococcus and Micromonospora, five same actinobacterial genera were found both in sponges and ascidians. In particular, the level of the diversity of ascidian-associated actinobacteria is similar to that of sponges, making ascidians a promising source of novel marine actinobacteria currently underexploited for the discovery of new bioactive marine natural products.