Pretreatment of lignocellulose for biochemical conversion commonly results in formation of by-products that inhibit microorganisms and cellulolytic enzymes. To make bioconversion processes more efficient, inhibition problems can be alleviated through conditioning. Ozone is currently commercially employed in pulp and paper production for bleaching, as it offers the desirable capability to disrupt unsaturated bonds in lignin through an ionic reaction known as ozonolysis. Ozonolysis is more selective towards lignin than cellulose, for instance, when compared to other oxidative treatment methods, such as Fenton's reagent. Ozone may thus have desirable properties for conditioning of pretreated lignocellulose without concomitant degradation of cellulose or sugars. Ozone treatment of SO2-impregnated steam-pretreated Norway spruce was explored as a potential approach to decrease inhibition of yeast and cellulolytic enzymes. This novel approach was furthermore compared to some of the most effective methods for conditioning of pretreated lignocellulose, i.e., treatment with alkali and sodium dithionite. Low dosages of ozone decreased the total contents of phenolics to about half of the initial value and improved the fermentability. Increasing ozone dosages led to almost proportional increase in the contents of total acids, including formic acid, which ultimately led to poor fermentability at higher ozone dosages. The decrease of the contents of furfural and 5-hydroxymethylfurfural was inversely proportional (R (2) > 0.99) to the duration of the ozone treatment, but exhibited no connection with the fermentability. Ozone detoxification was compared with other detoxification methods and was superior to treatment with Fenton's reagent, which exhibited no positive effect on fermentability. However, ozone detoxification was less efficient than treatment with alkali or sodium dithionite. High ozone dosages decreased the inhibition of cellulolytic enzymes as the glucose yield was improved with 13 % compared to that of an untreated control. Low dosages of ozone were beneficial for the fermentation of steam-pretreated Norway spruce, while high dosages decreased the inhibition of cellulolytic enzymes by soluble components in the pretreatment liquid. While clearly of interest for conditioning of lignocellulosic hydrolysates, future challenges include finding conditions that provide beneficial effects both with regard to enzymatic saccharification and microbial fermentation.