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The present study aimed to evaluate the antagonistic potential of Aspergillusspp. against Macrophomina phaseolina. All the tested species namely Aspergillus niger, Aspergillus terreus, Aspergillus flavipes, Aspergillus flavusand Aspergillus sydowiiwere identified by rDNA sequencing of ITS and β-tubulin regions prior to determine their antagonistic behaviour against the pathogen by using dual culture technique. A. flavipesproved to be the most effective in reducing the colony diameter of M. phaseolina(53%) followed by A. niger(41%) and A. flavus(37%) over control, whereas A. sydowiiand A. terreuswere the least effective with 6.2% and 5% in vitro biocontrol potentials, respectively. The secondary metabolites produced by A. flavipeswere tested against the genomic DNA of M. phaseolinato understand the mechanism of antagonism. The bioactive metabolites completely degraded the pathogenic fungal DNA at higher concentrations of 400% and 500% after 24 h. Moreover, the chloroform and ethyl acetate fractions of culture filtrates of A. flavipeswere subjected to GC–MS analysis for the identification of potential antimicrobial compounds and a total of 14 and 8 compounds were identified in these fractions, respectively. Among these 9,12-octadecadienoic acid (Z,Z)- followed by 1,2-benzedicarboxylic acid, diisooctyl ester; decane; hexadecanoic acid,2-hydroxy-1-(hydroxymethyl) ethyl ester; benzene, 1,2,3-trimethyl; n-hexadecanoic acid and naphthalene were the predominant compounds with peak areas of 31.72%, 26.27%, 18.47%, 10.62%, 6.82%, 6.04% and 5.67%, respectively. Literature shows that some of identified compounds possess antimicrobial properties, which might be responsible for control of M. phaseolina.