The RNase A superfamily lineage includes distant members with antimicrobial properties suggesting a common ancestral host defense role. In an effort to identify the minimal requirements for the eosinophil cationic protein (ECP or RNase 3) antimicrobial properties we have applied site directed mutagenesis on its closest family homologue, the eosinophil derived neurotoxin (EDN or RNase 2). Both eosinophil secretion proteins are involved in human immune defense, and are reported as among the most rapidly evolving coding sequences in primates. Previous studies in our laboratory defined two regions at the N-terminus involved in the protein antimicrobial action, encompassing residues 8 to 16 and 34 to 36. Here we demonstrate that switching two single residues is enough to provide EDN with ECP antipathogen properties. That is, the EDN double mutant Q34R/R35W displays an enhanced bactericidal activity, particularly towards Gram-negative bacteria, and a significant increase of its affinity towards the bacteria outer membrane lipopolysaccharides (LPS). Moreover, we confirmed the direct contribution of W35 residue in the LPS binding, membrane interaction and permeabilization processes. Furthermore, the additional T13 to I substitution provides EDN with an exposed hydrophobic patch required for the protein self-aggregation and triggers the bacteria agglutination, thereby increasing the final antimicrobial activity up to 20 fold. Our results highlight how single selected mutations can reshape the entire protein function. The present work provides an example of how structure guided protein engineering can successfully reproduce an evolution selection process towards the emergence of new physiological roles. This article is protected by copyright. All rights reserved.