The potential of using bio-based binders for medium density fiberboard (MDF) has received increased attention. However, no study was done on the potential of palm-based polyurethane (PU) as biobased binder in MDF. Therefore, an investigation on the effect of equivalent weight ratios of isocyanate (NCO) and hydroxyl group (OH) of palm oil-based PU binder and binder content on the properties of MDF was conducted. Palm oil-based PU binders were prepared at different equivalent weight ratios of NCO and OH at 1.5/1 (PU1.5), 2.0/1 (PU2.0) and 2.5/1 (PU2.5) and binder content at 3%, 5%, 7% and 10% were used in the preparation of MDF. The polyol, NCO and palm oil-based PU binders were characterised using Fourier transform infrared spectroscopy and thermogravimetric analysis. As equivalent weight ratios of NCO and OH and binder content increased, the modulus of rupture, modulus of elasticity and internal bonding of MDF were consistently increased. Thickness swelling and moisture content decreased with increased equivalent weight ratios of NCO and OH and binder content. In general, MDF prepared with PU2.0 with 7% binder content fulfills all British Standard (BS EN 622-5, 2009) requirements, indicating its suitability for MDF application.