The decline in antibiotic development, alongside the increasing prevalence of multidrug-resistant bacteria, highlights the urgent need for new antimicrobial agents. Among the pathogens prioritized by the World Health Organization for antimicrobial research and development are Staphylococcus aureus and Pseudomonas aeruginosa, which are associated with severe infections and significant resistance mechanisms that compromise treatment efficacy. In this context, bioprospecting has emerged as a promising strategy for identifying new antimicrobial compounds from diverse biological sources. This integrative literature review synthesized current methodologies used for the isolation, cultivation, processing, extraction, and identification of antibiotic-producing bacteria active against S. aureus and P. aeruginosa, providing an overview of approaches applied to pathogens of critical clinical relevance. The review was conducted using the MEDLINE portal through the PubMed database, and included in vitro studies published in English between 2020 and 2025 that aligned with the proposed objective, resulting in 44 eligible articles. The findings demonstrated that marine environments, soil, food sources, and the human microbiota are the primary sources of bacteria with antimicrobial potential against these pathogens. Lactic acid bacteria were the most frequently identified group, although other bacterial genera were also reported, such as Bacillus. Cultivation conditions varied according to the bacterial species and study objectives. Antibiotic substances were predominantly obtained through solvent extraction and ammonium sulfate precipitation, while structural characterization relied mainly on chromatographic techniques, nuclear magnetic resonance spectroscopy, and mass spectrometry. Overall, this review consolidates current methodological strategies in antimicrobial bioprospecting and may support future studies focused on the discovery and development of new antibacterial compounds targeting WHO priority pathogens.
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