Galactofuranose (Galf) is the five-membered ring form of galactose. It is widely distributed among several branches of the eukaryotic kingdom. This review highlights recent advances in our understanding of the biosynthesis and function of Galf-containing glycoconjugates in fungal Aspergillus spp. and the protozoan trypanosomatid parasites. We give an overview of the biosynthetic pathways leading to the production of glycolipids, glycoproteins and polysaccharides containing Galf in these species and their biological relevance. Remarkably, modification of the cell surface caused by Galf absence often results in morphological abnormalities and an impaired cell wall function in these organisms. Galf-deficient mutants are generally hypersensitive to drugs, exhibit a constitutive osmotic stress phenotype and/or have an attenuated virulence. Since Galf has never been found in mammals and higher plants, Galf-biosynthetic pathways have raised much interest as targets for drug development to combat microbial infections.