Five antibacterial peptides produced by Bacillus subtilis NCIB 3610 were purified, quantified, characterized, and identified in the present study. Cell-free extracts were subjected to three purification protocols employing ammonium sulfate or organic solvent precipitation and their combination, followed by ion-exchange chromatography, solid-phase extraction, and preparative high-performance liquid chromatography (HPLC). The combined ammonium sulfate and organic solvent precipitation extraction protocol presented the best results for peptide purification. In the five fractions that presented antimicrobial activity, antibacterial peptides were quantified by the turbidometric method and by HPLC using nisin for external calibration, with the second providing more accurate results. All peptides were pH- and temperature-resistant and their sensitivity to proteases treatment indicated their proteinic nature. The five peptides were subjected to microwave-assisted acid hydrolysis (MAAH) and following derivatization were analyzed using norleucine as the internal standard, to determine their amino acid content. The identification of the isolated peptides using the UniProt and PubChem databases indicated that the four peptides correspond to UniProt entries of the bacteriocins Subtilosin-A (Q1W152) Subtilosin-SbOX (H6D9P4), Ericin B (Q93GH3), Subtilin (P10946), and the fifth to the non-ribosomal antibacterial lipopeptide surfactin (CID:443592). The amino acid content determination and computational analyses, applied in the present work on the antimicrobial peptides of B. subtilis, proved an efficient screening and quantification method of bacteriocins that could potentially be applied in other bacterial strains. The constructed phylogenetic trees heterogeneity observed across the five peptides investigated might be indicative of competitive advantage of the strain.