Cellulosic ethanol production from biomass raw materials involves process steps such as pre-treatment, enzymatic hydrolysis, fermentation, and distillation. Use of streams within cellulosic ethanol production was explored for onsite enzyme production as part of a biorefinery concept. Sixty-four fungal isolates were in plate assays screened for lignocellulolytic activities to discover the most suitable fungal strain with efficient hydrolytic enzymes for lignocellulose conversion. Twenty-five were selected for further enzyme activity studies using a stream derived from the bioethanol process. The filter cake left after hydrolysis and fermentation was chosen as substrate for enzyme production. Five of the 25 isolates were further selected for synergy studies with commercial enzymes, Celluclast 1.5L and Novozym 188. Finally, IBT25747 (Aspergillus niger) and strain AP (CBS 127449, Aspergillus saccharolyticus) were found as promising candidates for onsite enzyme production where the filter cake was inoculated with the respective fungus and in combination with Celluclast 1.5L used for hydrolysis of pre-treated biomass.