Isoprene, synthesized through two complementary biosynthetic routes known as the mevalonate (MVA) pathway and the deoxy-xylulose phosphate pathway, is a valuable monomer that is used for rubber and several other chemical industries. Despite the recent interest in the industrial and biomedical applications of isoprene and its derivatives, the complexity of controlling its chemical synthesis due to the formation of greenhouse gases is a significant problem. To overcome the productivity and yield challenges, in addition to generating environmental and economic benefits, this study aimed to focus on the direct fermentation of cellulosic materials into bioisoprene. In this study, bioisoprene was synthesized via a biotransformation process through enzymatic hydrolysis of cassava peel using Aspergillus niger 11JK and Saccharomyces cerevisiae 19KB strain. The mevalonate (MVA) pathway (synthetic route) exploited during bioisoprene production by S. cerevisiae 19KB strain was investigated using the hydrolyzed cassava peel broth. The obtained crude extract was analyzed for bioisoprene yield and enzymatic activities using Gas chromatography. Furthermore, results of the size exclusion chromatography revealed the presence of polysaccharide hydrolyzing enzymes (e.g., amylase and cellulase), and mevalonate pathway enzymes, including isoprene synthase, mevalonate-5-diphosphate decarboxylase, and isopentyl phosphate kinase, in addition to isoprene, mevalonic acid (MVA), and its isomer dimethylallyl diphosphate (DMAPP). Based on the results obtained in this study, bioisoprene synthesis via direct fermentation of cheap and abundant carbon sources such as cassava peel using the S. cerevisiae 19KB strain will overcome the high production costs and low yield challenges of bioisoprene, thus generating significant environmental and economic benefits.