Pseudomonads are well-known for their plant growth-promoting properties and biocontrol capabilities against microbial pathogens. Recently, their potential to protect crops from parasitic plants has garnered attention. This study investigates the potential of different Pseudomonas strains to inhibit broomrape growth and to protect host plants against weed infestation. Four Pseudomonas strains, two P. fluorescens JV391D17 and JV391D10, one P. chlororaphis JV395B and one P. ogarae F113 were cultivated using various carbon sources, including fructose, pyruvate, fumarate, and malate, to enhance the diversity of potential Orobanche growth inhibition (OGI)-specialized metabolites produced by Pseudomonas strains. Both global and targeted metabolomic approaches were utilized to identify specific OGI metabolites. Both carbon sources and Pseudomonas genetic diversity significantly influenced the production of OGI metabolites. P. chlororaphis JV395B and P. ogarae F113 produced unique OGI metabolites belonging to different chemical families, such as hydroxyphenazines and phloroglucinol compounds, respectively. Additionally, metabolomic analyses identified an unannotated potential OGI ion, M375T65. This ion was produced by all Pseudomonas strains but was found to be over-accumulated in JV395B, which likely explains its superior OGI activity. Then, greenhouse experiments were performed to evaluate the biocontrol efficacy of selected strains: they showed the efficacy of these strains, particularly JV395B, in reducing broomrape infestation in rapeseed. These findings suggest that certain Pseudomonas strains, through their metabolite production, can offer a sustainable biocontrol strategy against parasitic plants. This biocontrol activity can be optimized by environmental factors, such as carbon amendments. Ultimately, this approach presents a promising alternative to chemical herbicides.