Antimicrobial peptides (AMPs) are natural antagonistic tools of many bacteria and are considered as attractive antimicrobial agents for the treatment of bacteria with multidrug resistance. Lactic acid bacteria from the gastrointestinal tract of animals and human produce various AMPs inhibiting the growth of pathogens. Here we report the isolation and identification of novel Lactobacillus fermentum strain HF-D1 from the human gut producing AMPs which prevents the growth of P. aeruginosa and S. marcescens. The active fraction of peptides was obtained from the culture liquid by precipitation at 80% saturation of ammonium sulphate. For peptides identification, the precipitate was treated with guanidine hydrochloride to desorb from proteins, separated with ultrafiltration on spin columns with 10,000 MWCO, desalted with a reversed-phase chromatography and subjected to LC–MS/MS analysis. The in silico analysis of the identified 1111 peptides by using ADAM, CAMPR3 and AMPA prediction servers led to identification of the linear peptide with highly probable antimicrobial activity and further investigation of its antibacterial activity mechanism is promising. By using the dereplication algorithm, the peptide highly similar to non-ribosomal cyclic AMPs originally isolated from Staphylococcus epidermidis has been identified. This indicates that L. fermentum HF-D1 represents a novel strain producing antimicrobial peptides targeting P. aeruginosa and S. marcescens.