Oil palm mesocarp fiber (OPMF), a biomass waste generated during the production of palm oil is rich in polysaccharides that can be converted to value-added product. The potential of cellulose from OPMF as a prebiotic represents an innovative exploration of biomass waste, which has never been undertaken. This study aims to investigate the effect of supplementation of OPMF in the medium on the growth of probiotic Saccharomyces cerevisiae, Lactobacillus casei and enteropathogenic E. coli, and to ascertain the potential OPMF as prebiotic by quantifying prebiotic activity score (PAS). The research was designed using a Randomized Complete Block Design with a single factor and three replications. The factor was the concentration of OPMF extract added to the growth medium, with seven treatment levels: P0 as control (no addition), P1 (1% glucose), P2 (2% prebiotic inulin), P3 (2% OPMF extract), P4 (4% OPMF extract), P5 (6% OPMF extract), P6 (8% OPMF extract), and P7 (10% OPMF extract) (w/v). The results showed that supplementation of OPMF extract significantly supported the growth of both of probiotics used in this study (p<0.05) similar to on the prebiotic inulin. The growth of S. cerevisiae was the highest on the 8% OPMF extract, with the PAS value of 1.90. In addition, the growth of L. casei on OPMF extract at the minimal concentration of 6% and on inulin were higher significantly than on glucose, with the PAS in the range of 1.98 – 2.47. In contrast, the growth of E. coli on the OPMF extract and on inulin were lower than on glucose (p<0.05). Conclusion, the growth of L. casei on OPMF extract was higher than S. cerevisiae, at a minimal concentration of 6%. Therefore, OPMF extract was potential as prebiotic.