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The climate-change-coupled fungal burden in crop management and the need to reduce chemical pesticide usage highlight the importance of finding sustainable ways to control <i>Aspergillus flavus</i>. This study examines the effectiveness of 50 <i>Pseudomonas</i> isolates obtained from corn rhizospheres against <i>A. flavus</i> in both solid and liquid co-cultures. The presence and quantity of aflatoxin B1 (AFB1) and AFB1-related compounds were determined using high-performance liquid chromatography–high resolution mass spectrometry analysis. Various enzymatic- or non-enzymatic mechanisms are proposed to interpret the decrease in AFB1 production, accompanied by the accumulation of biosynthetic intermediates (11-hydroxy-O-methylsterigmatocystin, aspertoxin, 11-hydroxyaspertoxin) or degradation products (the compounds C₁₆H₁₀O₆, C₁₆H₁₄O₅, C₁₈H₁₆O₇, and C₁₉H₁₆O₈). Our finding implies the upregulation or enhanced activity of fungal oxidoreductases and laccases in response to bacterial bioactive compound(s). Furthermore, non-enzymatic reactions resulted in the formation of additional degradation products due to acid accumulation in the fermented broth. Three isolates completely inhibited AFB1 or any AFB1-related compounds without significantly affecting fungal growth. These bacterial isolates supposedly block the entire pathway for AFB1 production in the fungus during interaction. Apart from identifying effective <i>Pseudomonas</i> isolates as potential biocontrol agents, this work lays the foundation for exploring new bacterial bioactive compounds.