A study was conducted to optimize sugar production from oil palm empty fruit bunch fiber (EFBF). Three different pretreatments were applied to EFBF, namely steam, steam with 5% sodium hydroxide (steam + 5% NaOH) and steam with 5% acetic acid (steam + 5% acetic acid). The hydrolyzability of the pretreated EFBF was determined using cellulolytic enzyme mixture comprising of Celluclast 1.5L (C), Viscozyme L (V) and Novozyme 188 (N). The different pretreatments showed varying degree of severities to the morphology of the fiber. Steam + 5% acetic acid pretreatment was found to cause the most severe changes to the EFBF surface. The optimum combinations of the enzymes for EFBF degradation, using a fixed parameters, were determined using Simplex Lattice mixture design. For untreated, steam pretreated, steam + 5% NaOH, steam + 5% acetic acid EFBFs, the optimum combinations enzyme were 0.49C: 0.45V: 0.06N, 0.88C: 0.12N, 0.78C: 0.03V: 0.2N and 0.90C: 0.03V: 0.07N respectively. Surface morphology of EFBF appears to be the major contributing factor that affects efficiency of enzymatic hydrolysis. Among these four samples, steam + 5% acetic acid pretreated EFBF gave the highest yield of sugars (44.36% xylose; 52.03% glucose). Applying the best enzymatic combinations, saccharification of pretreated samples was optimized using RSM. saccharification of the steam + 5% acetic acid pretreated EFBF was able to yield 99.90 +/- 10.92 mg g(-1) xylose and 281.77 +/- 28.00 mg g(-1) glucose at 2.51% enzyme loading (with total protein loading 22.1 mg per gram EFBF) in 4.55% EFBF for 35.08 h of reaction. Overall, through the combination of steam + 5% acetic acid pretreatment, enzyme mixture optimization and optimization of enzymatic saccharification, EFBF has the potential to produce substantial amount of sugars (62.36% xylose; 81.84% glucose). (C) 2013 Elsevier Ltd. All rights reserved.