In this study, we evaluated the protein expression changes in multidrug-resistant P. aeruginosa and C. albicans treated with recombinant defensin-d2 and actifensin from spinach and Actinomyces ruminicola, respectively, for 1 h. The proteome changes in the test organisms were analyzed by LC-MS/MS ESI using label-free data-dependent acquisition (DDA) quantitative proteomics technique. A total of 28 and 9 differentially expressed proteins (DEPs) were identified in the treated P. aeruginosa and C. albicans, respectively, with a 2-fold change threshold and p-value < 0.05. The GO and KEGG enrichment analysis found that DEPs related to binding, catalytic activity, cellular component organization and biogenesis, structural molecule activity and response to stimulus were significantly expressed in actifensin and defensin-treated P. aeruginosa, and the DEPs were mainly involved in DNA replication and repair, translation, membrane transport, energy metabolism, environmental adaptation, and signal transduction. DEPs in actifensin and defensin-treated C. albicans were related to cell growth and death, translation, lipid metabolism and energy metabolism were significantly expressed, and these DEPs mainly involved oxidative phosphorylation, RNA degradation, carbohydrate metabolism, and cell cycle pathways. We also established evidently through the different pathways affected, that the recombinant peptides exerted multiple mechanisms of action against the test organisms, sequentially or simultaneously.