The present study aims to evaluate the impact of extracts of yeast and Aspergillus niger as biotic elicitors, on flavonoids accumulation in callus culture of Silybum marianum. Also the gene expression levels of Chalcone synthase genes (CHS1, CHS2 and CHS3) which inter in flavonoids formation were evaluated by Quantitative realtime PCR (qRT-PCR) technique. The findings indicated that total flavonoids content got enhanced in response to elicitation by extract of yeast and A. niger in callus. Expression analysis of the three CHS genes in callus with elicitor treatments showed that biotic elicitors (yeast and A. niger extracts) strongly induced their expression. It was noted that the CHS2 expression was notably enhanced by A. niger and yeast extracts with 13.7- and 8.8-fold respectively compared to control. The results indicated that, biotic treatments contribute to the up regulation of CHS genes, which in turn, lead to an increase of flavonoids content in callus culture of S. marianum.