Co-culture is the simultaneous cultivation of two or more microorganisms in the same culture or growth medium. This technique allows observation and analysis of the interactions between microorganisms, such as competition for nutrients, cooperative behavior, or even the production of novel compounds that would not be synthesized by either species alone. This study aimed to use fungal co-culture for finding new sources of novel antifungals via a biological approach. In the current study, only 9 of 40 tested endophytic fungal isolates interacted positively with Fusarium proliferatum AUMC 15541by dual culture plate assay. Using well diffusion assay, extracts of Aspergillus ochraceus AUMC 15539 co-culture with F. proliferatum showed a greater inhibition action than monoculture. Large scale for Aspergillus-Fusarium co-culture (AF) was performed and its ethyl acetate extract exhibited significant inhibitory activity against different phytopathogens with potential effect against F. proliferatum (MIC = 6.25 mg/mL). The co-culture AF extract had LC50 values of 1972 (µg/mL) which is lower toxicity than single culture A and F. The total flavonoid, phenolic, and tannin contents of fungal extracts were significantly higher in the co-culture (AF) than in the single cultures A and F. High-performance liquid chromatography (HPLC) profiling of Aspergillus-Fusarium axenic and co-cultures (A), (F), and (AF) at different monitored wavelengths revealed the presence of a new peak at retention time 9.487 min in 235 nm chromatogram, in AF co-culture extract over A and F extracts.