Chaetomium globosum Kunze is recognized as a potential biocontrol fungus against spot blotch of wheat caused by Bipolaris sorokiniana. Its molecular mechanism of biocontrol activity and the biosynthetic pathways involved have not been yet elucidated. Here, global transcriptome profiling of C. globosum strain Cg2 during interaction with B. sorokiniana isolate BS112 using RNA-seq was performed in order to gain insights into the potential mechanisms of antagonism. The Illumina HiSeq platform (2 × 150 bp) yielded an average of 20–22 million reads with 50–58% GC. De novo assembly generated 45,582 transcripts with 27,957 unigenes. Transcriptome analysis displayed distinct expression profiles in the interaction (Cg2–BS112), out of which 6,109 unique differentially expressed genes were present. The predominant transcripts classified as genes involved in “catalytic activity” constituted 45.06%, of which 10.02% were associated with “hydrolytic activity” (GO:0008152), and similarly, in the biological process, 29.18% of transcripts were involved in “metabolic activity” (GO:0004096 and GO:0006979). Heat map and cluster categorization suggested an increase in the expression levels of genes encoding secondary metabolites like polyketide synthase (GO:0009058), S-hydroxymethyl glutathione dehydrogenase (GO:0006069), terpene cyclase (EC 4.2.3.-), aminotran_1_2 domain-containing protein (GO:0009058), and other hydrolytic CAZYmes such as the glycosyl hydrolase (GH) family (GH 13, GH 2, GH 31, and GH 81; GO:0005975), cellulase domain-containing protein, chitinases, β-1, 3-glucanases (GO:0004565), glucan endo-1,3-beta-glucanase (GO:0052861), and proteases (GO:0004177). The obtained RNA-seq data were validated by RT-qPCR using 20 randomly chosen genes, showing consistency with the RNA-seq results. The present work is worldwide the first effort to unravel the biocontrol mechanism of C. globosum against B. sorokiniana. It generated a novel dataset for further studies and facilitated improvement of the gene annotation models in the C. globosum draft genome.