Antimicrobial peptides are ubiquitous components of eukaryotic innate immunity. Defensins are a well-known family of antimicrobial peptides, widely distributed in ticks, insects, plants and mammals, showing activity against bacteria, viruses, fungi, yeast and protozoan parasites. Ixodes ricinus is the most common tick species in Europe and is a vector of pathogens affecting human and animal health. Recently, six defensins (including two isoforms) were identified in I. ricinus. We investigated the evolution of the antimicrobial activity of I. ricinus defensins. Among the five unique defensins, only DefMT3, DefMT5 and DefMT6 showed in vitro antimicrobial activity. Each defensin was active against rather distantly-related bacteria (P < 0.05), significantly among Gram-negative species (P < 0.0001). These three defensins represent different clades within the family of tick defensins, suggesting that the last common ancestor of tick defensins may have had comparable antimicrobial activity. Differences in electrostatic potential, and amino acid substitutions in the β-hairpin and the loop bridging the α-helix and β-sheet may affect the antimicrobial activity in DefMT2 and DefMT7, which needs to be addressed. Additionally, the antimicrobial activity of the γ-core motif of selected defensins (DefMT3, DefMT6, and DefMT7) was also tested. Interestingly, compared to full length peptides, the γ-core motifs of these defensins were effective against less species of bacteria. However, the antifungal activity of the γ-core was higher than full peptides. Our results broaden the scope of research in the field of antimicrobial peptides highlighting the overlooked ability of arthropod defensins to act against distantly-related microorganisms.