Present-day information available on the charophyte macroalgae in Egypt, including their phylogenetic affinities, remains largely incomplete. In this study, nine charophyte populations were collected from different aquatic biotopes across the Egyptian Western-Desert Oases and Sinai Peninsula. All populations were investigated using an integrative polyphasic approach including phylogenetic analyses inferred from the chloroplast-encoded gene (rbcL) and the internal transcribed spacer (ITS1) regions, in parallel with morphotaxonomic assignment, ultrastructure of the oospore walls, and autecology. The specimens identified belonged to the genera Chara, Nitella, and Tolypella, with predominance of the first genus to which five species were assigned though they presented some interesting aberrant taxonomic features: C. aspera, C. contraria, C. globata, C. tomentosa, and C. vulgaris. Based on our integrative study, the globally rare species C. globata was reported for the second time for the whole African continent. The genus Nitella was only represented by N. flagellifera, and based on the available literature, it is a new record for North Africa. Noteworthy, an interesting Tolypella sp., morphologically very similar to T. glomerata, was collected and characterized and finally designated with the working name ‘Tolypella sp. PBA–1704 from a desert, freshwater wetland’, mainly based on its concatenated rbcL+ITS1 phylogenetic position. This study not only improved our understanding on the diversity, biogeography and autecological preferences of charophytes in Egypt, but it also broadened our knowledge on this vulnerable algal group in North Africa, emphasizing the need of more in-depth research work in the future, particularly in the less–impacted desert habitats.