Marine cyanobacteria are a source of bioactive natural compounds, with a wide range of biotechnological applications. However, information on sponge-associated cyanobacteria are relatively scarce to date. In this paper, we carried out the morphological and molecular characterization of eight cyanobacterial strains, previously isolated from the Mediterranean sponge Petrosia ficiformis, and evaluated their biological activities on epithelial- and neuron-like cultured cells of human and murine origin. The new analysis allowed maintaining the assignment of three strains (Cyanobium sp., Leptolyngbya ectocarpi, and Synechococcus sp.), while two strains previously identified as Synechococcus sp. and Leptolyngbya sp. were assigned to Pseudanabaena spp. One strain, i.e., ITAC104, and the ITAC101 strain corresponding to Halomicronema metazoicum, shared extremely high sequence identity, practically representing two clones of the same species. Finally, for only one strain, i.e., ITAC105, assignment to a specific genus was not possible. Concerning bioactivity analyses, incubation of cyanobacterial aqueous cell supernatants induced variable responses in cultured cells, depending on cell type, with some of them showing toxic activity on human epithelial-like cells and no toxic effects on human and rat neuron-like cells. Future investigations will allow to better define the bioactive properties of these cyanobacteria strains and to understand if they can be useful for (a) therapeutic purpose(s).