Data from both the laboratory and clinic in the last decade indicate that antimicrobial peptides (AMPs) are widely regarded as potential sources of future antibiotics owing to their broad-spectrum activities, rapid killing, potentially low-resistance rate and multidirectional mechanisms of action compared to conventional antibiotics. Defensins, a prominent family of AMPs, have been found in a wide range of organisms including plants. Thailand is a rich source of plants including medicinal plants used therapeutically, however there is no report of defensin from among these plants. In this study, a novel plant defensin gene, BcDef, was successfully cloned from Brugmansia x candida (Bc). BcDef cDNA was 237 bp in length, encoding 78 amino acids with a putative 31-amino acid residue signal peptide at the N-terminal followed by the mature sequence. BcDef shared high sequence identity (78-85%) with Solanaceae defensins and belonged to the class I plant defensins. From homology modeling, BcDef shared a conserved triple stranded β-sheet (β1-β3) and one α-helix (α1) connected by a loop (L1-L3). BcDef1 peptide, designed from the γ-core motifs of BcDef located in loop 3, showed antibacterial activity against both Gram-positive and Gram-negative pathogens with the lowest MIC (15.70 μM) against Staphylococcus epidermidis. This peptide affected cell membrane potential and permeability, and caused cell membrane disruption. Moreover, BcDef1 also exhibited antioxidant activity and showed low cytotoxicity against mouse fibroblast L929 cells. These findings may provide an opportunity for developing a promising antibacterial agent for medical application in the future.