Plant defensins are a group of small disulfide-rich cationic peptides that exhibit a broad spectrum of antimicrobial activities. In the present study, an antibacterial plant defensin peptide was successfully identified and characterized from the transcriptome of the oat (Avena sativa L.), and called AsDef1. The complete nucleotide sequence of AsDef1 was determined (321 bp) and found to contain an open reading frame (ORF) encoding a peptide of 77 aa with a putative 22 aa signal peptide sequence that addresses the mature defensin to the apoplast. Further in silico analyses revealed that the structure of the identified defensin (AsDef1) consists of the Knot1 functional domain with eight conserved cysteine residues and four disulfide bonds. The highest expression of AsDef1 was observed in the developing seeds of the A. sativa plant. AsDef1 also showed antibacterial activity against both Gram-positive and Gram-negative bacteria. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values ranged from 0.15625 μM to 0.625 μM. In this study, we identified and characterized an antibacterial defensin from A. sativa for the first time. The findings of the present study offer insights that can be used in producing pathogen-resistant transgenic plants and in developing potential antibacterial agents in the future using AsDef1 from A. sativa.