Pseudomonas bacteria are renowned for their remarkable capacity to synthesize antibiotics, namely mupirocin, gluconic acid, pyrrolnitrin, and 2,4-diacetylphloroglucinol (DAPG). While these substances are extensively employed in agricultural biotechnology to safeguard plants against harmful bacteria and fungi, their potential for human medicine and healthcare remains highly promising for common science. However, the challenge of obtaining stable producers that yield higher quantities of these antibiotics continues to be a pertinent concern in modern biotechnology. Although the interest in antibiotics of Pseudomonas bacteria has persisted over the past century, many uncertainties still surround the regulation of the biosynthetic pathways of these compounds. Thus, the present review comprehensively studies the genetic organization and regulation of the biosynthesis of these antibiotics and provides a comprehensive summary of the genetic organization of antibiotic biosynthesis pathways in pseudomonas strains, appealing to both molecular biologists and biotechnologists. In addition, attention is also paid to the application of antibiotics in plant protection.