Among soil isolated species of actinomycetes bacteria that affected the growth of aflatoxin producing mold; Aspergillus flavus TISTR 3041, the isolate SP-O2 showed a promising inhibitory activity on the dual culture assay. This isolated bacterium was characterized on various International Streptomyces Project media. The macroscopic morphological analysis showed mycelium, sporogenesis, and orangish pink of colonial pigment. The scanning electron microscope (SEM) photograph showed that the sporogenesis has occurred by shrinking of both sides of aerial mycelium. After the sporogenesis has been completed, the fragmentation of altered mycelium started from the tips of aerial mycelium to produce 0.8-1 micrometer fragmented spores. The 16S rRNA sequences analysis revealed that isolate SP-O2 shared a 99.93 % (1 mismatch/1436 nt) similarity with Streptomyces spectabilis NBRC 13424 AB184393. Co-culture liquid assay was analyzed for the aflatoxin produced by A. flavus in the presence of Streptomyces spectabilis SP-O2. The result showed that UV-visualized band of aflatoxin B1 was not detected by co-cultivation of Streptomyces spectabilis SP-O2 with A. flavus by thin layer chromatography compared to the control. Indeed, fungal growth of A. flavus was destroyed by the antifungal substance produced by Streptomyces spectabilis SP-O2 in the liquid culture. Furthermore, biocontrol of contaminated A. flavus was conducted on peanut kernels and the results showed that Streptomyces spectabilis SP-O2 has antifungal activity against the presence of A. flavus. Together, these results suggested that further investigation on the development of biocontrol use of Streptomyces spectabilis SP-O2 to overcome the contamination of aflatoxin producing mold in the feedstock and agricultural-based industry is needed. HIGHLIGHTS The isolated actinobacteria in this study was identified as Streptomyces spectabilis isolate SP-O2 based on its morphological characteristics, physiological properties and molecular identification SEM showed the sporogenesis of 0.8-1 micrometer fragmented spores Streptomyces spectabilis isolate SP-O2 exhibited the promising fungicidal activity against flavus in the dual culture agar assay, co-culture liquid assay and peanut kernels assay with consistency results The secreted metabolites play a crucial role on the fungal killing against Aspergillus flavus rather than the aflatoxin degradation GRAPHICAL ABSTRACT