Healthcare-associated infections (HAIs) pose significant challenges to global health due to pathogen complexity and antimicrobial resistance. Biosensors utilizing antimicrobial peptides offer innovative solutions. Hylarana picturata Multiple Active Peptide 1 (Hp-MAP1), derived from Temporin-PTA, exhibits antibacterial properties sourced from the skin secretions of the Malaysian fire-bellied frog. An innovative sensing layer was developed for the electrochemical biorecognition of diverse pathogens: Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, and Staphylococcus aureus. Electrochemical impedance spectroscopy differentiated microorganisms based on distinct electrochemical responses. The sensor layer, composed of functionalized multi-walled carbon nanotubes (MWCNTs) associated with Hp-MAP1, exhibited varying levels of charge transfer resistance (R