Antimicrobial peptides (AMPs) have emerged as promising therapeutic alternatives to fight against the diverse infections caused by different pathogenic microorganisms. In this context, theoretical approaches in bioinformatics have paved the way toward the creation of several in silico models capable of predicting antimicrobial activities of peptides. All current models have several significant handicaps, which prevent the efficient search for highly active AMPs. Here, we introduce the first multitarget (mt) chemo-bioinformatic model devoted to performing alignment-free prediction of antibacterial activity of peptides against multiple Gram-positive bacterial strains. The model was constructed from a data set containing 2488 cases of AMPs sequences assayed against at least 1 out of 50 Gram-positive bacterial strains. This mt-chemo-bioinformatic model displayed percentages of correct classification higher than 90.00% in both training and prediction (test) sets. For the first time, two computational approaches derived from basic concepts in genetics and molecular biology were applied, allowing the calculations of the relative contributions of any amino acid (in a defined position) to the antibacterial activity of an AMP and depending on the bacterial strain used in the biological assay. The present mt-chemo-bioinformatic model constitutes a powerful tool to enable the discovery of potent and versatile AMPs.