Cryoconite holes are supraglacial depressions containing water and microbe-mineral aggregates. Their autotrophic component plays a central role in reducing the albedo of glaciers and could contribute to sustaining the cryoconite food web. However, knowledge of its diversity is still limited, especially in Antarctica. Moreover, the study of cryoconite microalgae is challenging due to the limitations of molecular approaches, such as incomplete genetic databases and the semiquantitative nature of the data. Furthermore, it is equally difficult to examine the development of microalgae in sediment by using standard counting methods for water-living organisms. By using an adaptation of the high-speed density gradient centrifugation method, we provide a comprehensive description of the phenotypic characteristics, abundance and community structure of microalgae and Cyanobacteria in different cryoconite holes located in different glaciers of Northern Victoria Land, East Antarctica. We described 36 morphotypes belonging to Cyanobacteria, green algae and diatoms, revealing that cryoconite holes encompass a remarkably high diversity of photoautotrophs. The adapted protocol enabled the application of a standard microscopic approach, which provided crucial and comparable information on morphological characteristics, biovolume and community organization from a unique environment. The study poses the basis for the taxonomy of photoautotrophs as well as their diversity and distribution in cryoconite habitats.