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 (RCT) for different microorganisms. Gram-negative species, especially P. aeruginosa, displayed higher RCT values, indicating better impedimetric responses. Excellent LODs were observed for P. aeruginosa (0.60), K. pneumoniae (0.42), E. coli (0.67), and S. aureus (0.59), highlighting the efficacy of the MWCNTs/Hp-MAP1 biosensor in microbial identification. The MWCNTs/Hp-MAP1 biosensor platform presents a promising and effective microbial identification strategy with potential healthcare applications to mitigate HAIs and enhance patient care.