With the growing demand for new antibiotics to combat increasing multi-drug resistance, a family of antimicrobial peptides known as cathelicidins has emerged as potential candidates. Expansions in cathelicidin-encoding genes in marsupials and monotremes are of specific interest as the peptides they encode have evolved to protect immunologically naive young in the harsh conditions of the pouch and burrow. Our previous work demonstrated that some marsupial and monotreme cathelicidins have broad-spectrum antibacterial activity and kill resistant bacteria, but the activity of many cathelicidins is unknown. To investigate associations between peptide antimicrobial activity and physiochemical properties, we tested 15 cathelicidin mature peptides from tammar wallaby, grey short-tailed opossum, platypus and echidna for antimicrobial activity against a range of bacterial and fungal clinical isolates. One opossum cathelicidin ModoCath4, tammar wallaby MaeuCath7 and echidna Taac-CATH1 had broad-spectrum antibacterial activity and killed methicillin-resistant Staphylococcus aureus. However, antimicrobial activity was reduced in the presence of serum or whole blood, and non-specific toxicity was observed at high concentrations. The active peptides were highly charged, potentially increasing binding to microbial surfaces, and contained amphipathic helical structures, which may facilitate membrane permeabilisation. Peptide sequence homology, net charge, amphipathicity and alpha helical content did not correlate with antimicrobial activity. However active peptides contained a significantly higher percentage of cationic residues than inactive ones, which may be used to predict active peptides in future work. Along with previous studies, our results indicate that marsupial and monotreme cathelicidins show potential for development as novel therapeutics to combat increasing antimicrobial resistance.