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Waste glycerol is the main by-product generated during biodiesel production, in an amount reaching up to 10% of the produced biofuel. Is there any method which allows changing this waste into industrial valuable compounds? This manuscript describes a method for valorization of crude glycerol via microbial bioconversion. It has been shown that the use of free and immobilized biocatalysts obtained from <i>Gluconobacter oxydans</i> can enable beneficial valorization of crude glycerol to industrially valuable dihydroxyacetone. The highest concentration of this compound, reaching over 20 g&#183;L^&#8722;1, was obtained after 72 h of biotransformation with free <i>G. oxydans</i> cells, in a medium containing 30 or 50 g&#183;L^&#8722;1 of waste glycerol. Using a free cell extract resulted in higher concentrations of dihydroxyacetone and a higher valorization efficiency (up to 98%) compared to the reaction with an immobilized cell extract. Increasing waste glycerol concentration to 50 g&#183;L^&#8722;1 causes neither a faster nor higher increase in product yield and reaction efficiency compared to its initial concentration of 30 g&#183;L^&#8722;1. The proposed method could be an alternative for utilization of a petrochemical waste into industry applicated chemicals.