Vertebrate secreted RNases (ribonucleases) are small proteins that play important roles in RNA metabolism, angiogenesis or host defence. In the present study we describe the antimicrobial properties of the N-terminal domain of the hcRNases (human canonical RNases) and show that their antimicrobial activity is well conserved among their lineage. Furthermore, all domains display a similar antimicrobial mechanism, characterized by bacteria agglutination followed by membrane permeabilization. The results of the present study show that, for all antimicrobial hcRNases, (i) activity is retained at the N-terminus and (ii) the antimicrobial mechanism is conserved. Moreover, using computational analysis we show that antimicrobial propensity may be conserved at the N-terminus for all vertebrate RNases, thereby suggesting that a defence mechanism could be a primary function in vertebrate RNases and that the N-terminus was selected to ensure this property. In a broader context, from the overall comparison of the peptides' physicochemical and biological properties, general correlation rules could be drawn to assist in the structure-based development of antimicrobial agents.