Enzymatic bio-processing of cotton generates significantly less hazardous, readily biodegradable wastewater effluents, yet having several critical shortcomings, such as expensive processing costs and slow reaction rates, which impedes its acceptance at an industrial level. Our research showed that the introduction of a low-energy, uniform ultrasound field in enzyme-processing solutions greatly improved enzyme efficiency by significantly increasing their reaction rate. It has been established that the following specific features of combined enzyme/ultrasound bio-processing of cotton are critically important: a) the cavitation effects caused by the introduction of an ultrasound field in the enzyme processing solution greatly enhance the transport of enzyme macromolecules towards the substrate surface, b) the mechanical impact, produced by the collapse of the cavitation bubbles, provides an important benefit, that of “opening up” the surface of the solid substrates to the action of enzymes, c) the effect of cavitation is several hundred times higher in heterogeneous (solid substrate-liquid) than in homogeneous systems, and d) in water, the maximum effects of cavitation occur at ~50 °C, which is the optimum temperature for many industrial enzymes. At a laboratory scale, the introduction of ultrasonic energy in the reaction chamber during enzymatic biopreparation of greige cotton fabrics and enzymatic bio-conversion of cotton gin and cotton lint waste biomass in sugars resulted in a significant improvement in enzyme efficiency.