The increasing number of multidrug resistance microorganisms is an alarming threat, and their rapid detection is essential to prevent nosocomial, foodborne, or waterborne infections. Many peptides derived from the venom of wasp Synoeca surinama have antimicrobial activity against Gram-positive and Gram-negative bacteria. Synoeca-MP, an antimicrobial peptide (AMP) from mastoparan family, seems to increase bacterial membrane permeability, promoting cytotoxicity and membrane disruption. Here Synoeca-MP was evaluated as biorecognition element tethered over chitosan-coated magnetic nanoparticles (Fe3O4-Chit). The transducing layer of the biosensor was developed from the self-assembling of 4-mercaptobenzoic acid (4-MBA) monolayer onto gold substrate. Atomic force microscopy (AFM) analyses confirmed the biointeraction between AMP and different pathogens membranes. The fabrication and performance of the biosensing assembly were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Detection of Enterococcus faecalis (G+), Klebsiella pneumoniae (G-), Pseudomonas aeruginosa (G-), and Candida tropicalis was assessed in a recognition range from 101 to 105 CFU.mL−1. An instrumental limit of detection of 10 CFU.mL−1 was obtained for each specimen. However, the device presented a preferential selectivity towards Gram-negative bacteria. The proposed biosensor is a sensitive, fast, and straightforward platform for microbial detection in aqueous samples, envisaged for environmental monitoring applications. •A new electrochemical biosensor with high sensitivity towards Gram-negative species is presented.•The biosensor combines chitosan-coated magnetic nanoparticles with the antimicrobial peptide Synoeca-MP.•The sensor detected bacteria and fungi species with a LOD of 101 CFU mL−1.•The platform presented high sensitivity towards K. pneumoniae.•This biosensor stands out as a promising tool for fast microbial detection in liquid samples.