Trichoderma harzianum EU2-77 was a mutant strain of the wild-type strain T. harzianum NP13a isolated in Singapore. A multi-mutagenesis one-screening (MMOS) method was developed to further improve strain EU2-77 and a new mutant EUA20 was obtained. It exhibited filter paper cellulase (FPase) activity up to 14.79 IU/mL within 6 days shake flask cultivation. Activities of FPase, endoglucanase, beta-glucosidase, and xylanase, and protein content by EUA20 were respectively increased to 5.73, 4.35, 7.34, 1.80 and 2.70 folds. Using pretreated oil palm empty fruit bunch (OPEFB) and corncob powder as the substrates, strain EUA20 presented approximate 6.52 and 8.80 IU/ml FPase activity. Reducing sugar yield of 615.8 and 636.8 mg/g biomass were respectively obtained for OPEFB and corncob powder using cellulolytic enzymes of strain EUA20. Our results demonstrated that mutant strain EUA20 had great potential in on-site cellulase production for effective biomass bioconversion. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.