Background: The rise of microbial antibiotic resistance is a leading threat to the health of the human population. As such, finding new approaches to tackle these microbes, including development of novel antibiotics is vital. Results: In this study, we mined a rumen eukaryotic metatranscriptomic library for novel Antimicrobial peptides (AMPs) using computational approaches and thereafter characterised the therapeutic potential of the AMPs. We identified a total of 208 potentially novel AMPs from the ruminal eukaryotome, and characterised one of those, namely Lubelisin. Lubelisin (GIVAWFWRLAR) is an α-helical peptide, 11 amino acid long with theoretical molecular weight of 1373.76 D. In the presence of Lubelisin, strains of methicillin-resistant Staphylococcus aureus (MRSA) USA300 and EMRSA-15 were killed within 30 min of exposure with ≥103 and 104 CFU/mL reduction in viable cells respectively. Cytotoxicity of Lubelisin against both human and sheep erythrocytes was low resulting in a therapeutic index of 0.43. Membrane permeabilisation assays using propidium iodide alongside transmission electron microscopy revealed that cytoplasmic membrane damage may contribute to the antimicrobial activities of Lubelisin. Conclusions: We demonstrate that the rumen eukaryotome is a viable source for the discovery of antimicrobial molecules for the treatment of bacterial infections and further development of these may provide part of the potential solution to the ongoing problem of antimicrobial resistance. The role of these AMPs in the ecological warfare within the rumen is also currently unknown.