<p>Biological control using fungi holds significant potential for managing pests and pathogens, especially when different agents are applied together. Several studies have investigated the interaction of fungi, focusing on competition and antibiosis in simple, single-pathogen systems. This neglects how spatial and temporal dynamics profoundly influence outcomes in complex host ecosystems. Using a 2D Petri dish system, this study investigated the impact of initial spatial positioning on interactions among key fungal biocontrol agents (<i>Trichoderma</i>, <i>Metarhizium</i>, and <i>Beauveria</i>) and the pathogen <i>Fusarium oxysporum</i>. Different fungal isolates were line-streaked on potato dextrose agar in various combinations and arrangements. Each fungal growth area in Petri dish was calculated by ImageJ after the incubation period and the interactions between them were assessed. In the pairwise interactions, <i>T. afroharzianum</i>, <i>T. guizhouense</i>, and <i>M. brunneum</i> isolates all demonstrated strong antagonism, consistently outcompeting <i>F. oxysporum</i>. <i>Beauveria bassiana</i>, however, was the least competitive, showing little antagonism against the pathogen. In three-way interactions, the initial arrangement of the fungi significantly influenced competitive outcomes, which may be isolate- or strain-specific. The antagonistic effects of <i>Trichoderma</i> were modulated by the presence and position of <i>Metarhizium</i> and <i>Beauveria</i>, highlighting that multi-way interactions cannot be predicted from pairwise interactions alone. Our results underscore the importance of initial spatial positioning, providing an essential basis for optimizing application methods and designing more effective, location-dependent microbial consortia.</p>