The effects of the morphology and conformations of the surface biopolymers present on lignocellulosic biomass as well as their steric hindrance on enzymatic adsorption to biomass surfaces remain elusive. In a step to better understand these effects, nanoscale steric forces between a model surface that represents the hydrophobic residues of a cellulase enzyme and a set of reference lignocellulosic substrates were measured using atomic force microscopy (AFM) in liquid media. The reference substrates investigated were prepared by kraft, sulfite, and organosolv pulping pretreatment methods and varied in their surface lignin, xylan, and acetone extractives' contents. Measured steric forces were quantified through fitting to a model developed to describe polyelectrolytes brushes in terms of a brush thickness and a brush grafting density. Our data indicated that cellulose microfibrils extend from the microfibril matrix leading to a long-range steric repulsion and low attractive forces, to the hydrophobic model of the enzyme, suggesting that steric hindering can be a possible mechanism for nonproductive binding of enzymes to cellulose. When the amount of xylan increased in the absence of lignin, steric repulsions between the hydrophobic model of the enzyme, and biomass biopolymers decreased as.a result of collapsed cellulose microfibrils and adhesion forces increased. This suggests that leaving a small amount of xylan after biomass pretreatment can help improve enzymatic binding to cellulose. Irrespective of the type of lignin present on biomass, grafting densities increased and brush thicknesses decreased compared to those of lignin-free substrates. When compared to lignin-free substrates, lignin-containing substrates had higher attractive forces and lower steric repulsive forces. In addition, AFM images of the reference substrates in the wet and dry states showed that lignin precipitates on the biomass surface where kraft lignin had the highest particle size leading to a limited accessibility of the enzyme to the cellulose in biomass. When the effects of lignin precipitate size, the adhesion force, and steric forces on nonproductive enzymatic binding were all considered, our results indicate that organosolv pretreatment should be the treatment of choice to minimize enzymatic nonproductive binding to lignin.