Capsicum spp. (hot peppers) demonstrate a range of interesting bioactive properties spanning anti-inflammatory, antioxidant, and antimicrobial activities. While several species within the genus are known to produce antimicrobial peptides (AMPs), AMP sequence mining of genomic data indicates this space remains largely unexplored. Herein, in silico AMP predictions were paired with peptidomics to identify novel AMPs from the interspecific hybrid ghost pepper (Capsicum chinense × frutescens). AMP prediction algorithms revealed 115 putative AMPs within the Capsicum chinense genome, of which 14 were identified in the aerial tissue peptidome. PepSAVI-MS, de novo sequencing, and complementary approaches were used to fully molecularly characterize two novel AMPs, CC-AMP1 and CC-AMP2, including elucidation of a pyroglutamic acid post-translational modification of CC-AMP1 and disulfide bond connectivity of both. Both CC-AMP1 and CC-AMP2 have little homology with known AMPs and exhibited low μM antimicrobial activity against Gram-negative bacteria, including Escherichia coli. These findings demonstrate the complementary nature of peptidomics, bioactivity-guided discovery, and bioinformatics-based investigations to characterize plant AMP profiles.