Ethanol is one of the most important biofuels that can be produced from different renewable sources. Molasses and Venasses are used as cheap raw materials in the isolation of microorganisms and used molasses as renewable materials for ethanol production. Molasses and Venasses are considered important by-products in the sugar industry. This study aims to isolate and identify yeasts and bacteria present in both molasses and venasses to use them in the production of bioethanol. Molasses and venasses samples were collected from ten different sugar factories (Guirga, Savola, Deshna, Komombo, Abokorkaus, Delta, Dakahlia, Qus, Nag-hamdy, Armant) and were used to isolate different microorganisms that were screened for their bioethanol productivity. The results showed that the molasses samples contained more microbes than venasses. Twelve isolates were molecularly identified as S. cerevisiae by PCR-specific primers, while 64 isolates were bacterial isolates. All the yeast and bacterial isolates were screened for bioethanol productivity. Isolate M3 showed the highest bioethanol productivity (74%) and was identified by 16S rRNA gene sequencing as Klebsiella pneumoniae. Several factors affected the production of bioethanol, including sugar concentration, urea, and ammonium sulfate. When molasses was used as the carbon source, Klebsiella pneumoniae produced 1% (v/v) bioethanol by utilizing 20% molasses (sugar concentration), 0.4% urea, and 0.4% ammonium sulfate. When UV- mutagenesis was used to improve the bioethanol productivity, all the obtained mutants showed lower productivity compared to the wild-type (M3 isolate).