The multiprotein-bridging factor 1 (MBF1) gene family is well known in archaea, non-lichenized fungi, plants, and animals, and contains stress tolerance-related genes. Here, we identified four unique mbf1 genes in the lichenized fungi Endocarpon spp. A phylogenetic analysis based on protein sequences showed the translated MBF1 proteins of the newly isolated mbf1 genes formed a monophyletic clade different from other lichen-forming fungi and Ascomycota groups in general, which may reflect the evolution of the biological functions of MBF1s. In contrast to the lack of function reported in yeast, we determined that lysine114 in the deduced Endocarpon pusillum MBF1 protein (EpMBF1) had a specific function that was triggered by environmental stress. Further, the Endocarpon-specific C-terminus of EpMBF1 was found to participate in stress tolerance. Epmbf1 was induced by a number of abiotic stresses in E. pusillum and transgenic yeast, and its stress-resistant ability was stronger than that of the yeast mbf1. These findings highlight the evolution and function of EpMBF1 and provide new insights into the co-evolution hypothesis of MBF1 and TATA-box-binding proteins.