The production of value added chemicals from the waste stream of the biodiesel industry represents a feasible way to overcome the roadblocks this sector is currently facing. The surplus glycerol, the main by-product of the biodiesel manufacturing process, has become a liability for the industry, leading to a growing research on its use as feedstock in biological, chemical, and physical processes for the production of value-added products. In this research, we pre-treated glycerol with ozone, and investigated the effect on ethanol production through bacterial fermentation. A minimal medium was developed during preliminary studies and selected as substrate, and two different ozonation times and pH conditions were tested. Glycerol concentration was set at 10 g/L. Growth parameters resulted in slightly higher OD 600 values in the non-ozonated substrates than in the ozonated ones, reaching a maximum of 0.2 and 0.14 at pH value of 6.3 and 7.0, respectively. However, higher ethanol production resulted in the ozonated substrates. Ozonation seemed to actually enhance ethanol production at neutral pH and shorter ozonation time, 30 min instead of 120 min. This represents an encouraging result, considering shorter ozonation times require less energy input and produce higher ethanol yields. Ethanol production at these experimental conditions showed the highest difference between the two substrates, increasing from an average of 0.61 g/L in the fermenters with non-pre-treated glycerol, to an average of 0.81 g/L in the ones with ozonated glycerol. Energy input/gain analysis showed that the process is still not beneficial in terms of net energy gained, but break-even point was closed enough to suggest a possible optimization. Subsequently, we supplemented the glycerol-based medium with decreasing amount of the rich nutrients yeast extract and triptone. Growth parameters for both the fermenters with ozonated and non-ozonated glycerol was significantly lower compared to results reported in literature. Although the fermenters with the supplemented non-ozonated glycerol obtained higher ethanol production compared to the ones with non-ozonated glycerol, ozonation process seemed not to increase ethanol production in rich media. This suggests that future experiments on the potential of the ozonation process should focus on the minimal medium tested in this research.