Arabidopsis thaliana is a model plant for genomic studies. Nevertheless, much more work needs to be done to correlate all the genes present in its genome with protein function. This work reports the isolation and characterization of peptides from different Arabidopsis thaliana ecotypes CS1084, CS1298, CS1308, CS1566, CS1438, CS1138, CS1194, CS920, CS1643, CS1198 and CS1438 with antimicrobial activity and similarities to plant defensins. These peptides from several ecotypes were purified using a Red-Sepharose Cl-6B affinity column followed by a reversed-phase HPLC (Vydac C18-TP). Molecular masses were estimated by SDS-PAGE, where a major band with less than 10 kDa was observed. In vivo bioassays clearly showed that ecotype CS1643 was able to cause deleterious effects against E. coli, a gram-negative bacteria. On the other hand, no inhibitory effect was observed toward the filamentous fungi Aspergillus flavus, Rhizoctonia solani and Fusarium oxysporum and also against the gram-positive bacteria Bacillus subtilis. Because there are several genes corresponding to proteins from defensin family in the Arabidopsis genome, we expect to obtain a primary structure with high identity to this protein group. This research will possibly open new opportunities for the development through biotechnology of a novel antibacterial compound with low collateral effects. Further experiments will determine whether these peptides also have an effect against phytopathogens.