The saline-alkaline crater-lake Dziani Dzaha (Mayotte, Indian Ocean) is dominated by the bloom-forming cyanobacterium Arthrospira. However, the rest of the phototrophic community remains underexplored because of their minute dimension or lower biomass. To characterize the phototrophic microorganisms living in this ecosystem considered as a modern analog of Precambrian environments, several strains were isolated from the water column and stromatolites and analyzed using the polyphasic approach. Based on morphological, ultrastructural and molecular (16S rRNA gene, 18S rRNA gene, 16S-23S internal transcribed spacer (ITS) region and cpcBA-IGS locus) methods, seven filamentous cyanobacteria and the prasinophyte Picocystis salinarum were identified. Two new genera and four new cyanobacteria species belonging to the orders Oscillatoriales (Desertifilum dzianense sp. nov.) and Synechococcales (Sodalinema komarekii gen. nov., sp. nov., Sodaleptolyngbya stromatolitii gen. nov., sp. nov. and Haloleptolyngbya elongata sp. nov.) were described. This approach also allowed to identify Arthrospira fusiformis with exclusively straight trichomes instead of the spirally coiled form commonly observed in the genus. This study evidenced the importance of using the polyphasic approach to solve the complex taxonomy of cyanobacteria and to study algal assemblages from unexplored ecosystems.