ABSTRACT To decipher the dispersal range of the cosmopolitan green microalga Tetracystis vinatzeri, two airborne-collected strains were barcoded using the 18S ribosomal DNA gene (18S) and the mitochondrial cytochrome c oxidase I subunit (COI), and their survival capacities were investigated across a stepwise acidic to alkaline pH gradient. Genetic fingerprinting confirmed the identity of the strains as representing the same biological species. Investigations of the tolerance of T. vinatzeri to pH levels refined the delimitation of its ecological niche. T. vinatzeri showed optimal growth in freshwater habitats at pH 4.3–9.6 and strains could cope with exposure to slightly more acidic (down to 3.8) and alkaline (up to 10.4) conditions. However, organismal survival was drastically affected by exposure to pH < 3 and > 11. Morphological observations showed that T. vinatzeri could better cope with exposure to acidic pH, exhibiting less cell membrane damage. Genetic analysis showed that the two airborne strains shared the same haplotype, suggesting that they either originated from the same air mass or that the haplotype has a widespread distribution. Further phylogeographic and physiological investigations are required to define the spreading dynamic of T. vinatzeri.