Peptides and proteins with antimicrobial activity are produced throughout all kingdoms in nature, from prokaryotes to lower and higher eukaryotes, including fungi, plants, invertebrates and vertebrates. These proteins contribute to an important constitutive or induced defense mechanism of the producer against microorganisms. According to their variety in structure and function, these proteins are classified arbitrarily into groups that are based on their mechanism of action, their structure and their similarity to other known proteins. The present review focuses on a new group of antimicrobial proteins, namely small, basic and cysteine-rich antifungal proteins, which are secreted from filamentous fungi of the group Ascomycetes. These proteins are encoded by orthologous genes and exhibit both similarities and differences concerning their species-specificity, primary structure, protein activity and target sites. The properties of these proteins, their possible mode of action and their potential application for human benefits are discussed in comparison with other already well known antimicrobial proteins.