Hydrophilins are proteins that occur in all domains of life and protect cells and organisms against drought and other stresses. They include most of the Late Embryogenesis Abundant (LEA) proteins and the heat shock protein Hsp12. Here, the role of a predicted LEA-like protein (LeamA) and two Hsp12 proteins (Hsp12A, Hsp12B) of Neosartorya fischeri was studied. This filamentous fungus forms ascospores that belong to the most stress-resistant eukaryotic cells described to date. Heterologous expression of LeamA, Hsp12A, and Hsp12B resulted in increased tolerance against salt and osmotic stress in Escherichia coli. These proteins were also shown to protect lactate dehydrogenase against dry heat and freeze-thaw cycles in vitro. Deletion of leamA caused diminished viability of sexual ascospores after drought and heat. This is the first report on functionality of Hsp 12 and putative LeamA proteins derived from filamentous fungi, and their possible role in N. fischeri ascospore resistance against desiccation, high temperature and osmotic stress is discussed.