Hemicellulose was recovered from oil palm empty fruit bunch (EFB) fiber and used for producing xylooligosaccharides (XOS) prebiotics. EFB fibers were treated sequentially with peracetic acid (PA) and alkaline peroxide (AP). The PA treatment removed ∼50 % lignin. The subsequent AP treatment recovered the hemicellulose. The AP treatment was optimized using Taguchi experiment design with L16 orthogonal array consisting of 5 factors (NaOH concentration, H2O2 concentration, MgSO4 concentration, incubation temperature, treatment time) and 4 levels. Data from 16 experimental runs showed that the main effect of the factors on percentage hemicellulose recovery was in the order: NaOH > MgSO4 > incubation temperature > time > H2O2. The optimal conditions for hemicellulose isolation were: 15 % w/v NaOH in the reaction system (3 g NaOH/g delignified EFB), 1% w/v MgSO4 (0.2 g MgSO4/g delignified EFB), 45 °C incubation temperature and a 3 h reaction time. The PA-AP treatments solubilized nearly 53 % of the original dry mass of EFB. The enriched hemicellulose yield on EFB was 18.4 % by weight. Acid hydrolysis of the released hemicellulose produced xylose (44.1 %), glucose (17.6 %), arabinose (2.2 %), galactose (2.2 %), and mannose (4.4 %). Thermomyces lenuginosus xylanase (294.4 U L−1) was used to produce XOS from hemicellulose at 55 °C in a 12-h reaction. The XOS were assessed as substrates for two probiotic bacteria found in human gastrointestinal tract. The probiotic Lactobacillus plantarum WU-P19 utilized XOS better than Bifidobacterium bifidum TISTR 2129. Coculture of the two bacteria was shown to produce mixed fatty acids.