In response to the escalating demand for sustainable agricultural methodologies, the utilization of microbial volatile organic compounds (VOCs) as antagonists against phytopathogens has emerged as a viable eco-friendly alternative. Microbial volatiles exhibit rapid diffusion rates, facilitating prompt chemical interactions. Moreover, microorganisms possess the capacity to emit volatiles constitutively, as well as in response to biological interactions and environmental stimuli. In addition to volatile compounds, these bacteria demonstrate the ability to produce soluble metabolites with antifungal properties, such as APE Vf, pyoverdin, and fragin. In this study, we identified two Pseudomonas strains (BJa3 and MCal1) capable of inhibiting the in vitro mycelial growth of the phytopathogenic fungus Aspergillus flavus, which serves as the causal agent of diseases in sugarcane and maize. Utilizing GC/MS analysis, we detected 47 distinct VOCs which were produced by these bacterial strains. Notably, certain volatile compounds, including 1-heptoxydecane and tridecan-2-one, emerged as primary candidates for inhibiting fungal growth. These compounds belong to essential chemical classes previously documented for their antifungal activity, while others represent novel molecules. Furthermore, examination via confocal microscopy unveiled significant morphological alterations, particularly in the cell wall, of mycelia exposed to VOCs emitted by both Pseudomonas species. These findings underscore the potential of the identified BJa3 and MCal1 Pseudomonas strains as promising agents for fungal biocontrol in agricultural crops.