The rapid increase in biofuels world consumption has generated a glycerol surplus of about to 10 wt% of the overall biodiesel. With Colombia as one of the principals Latin America biodiesel producer, with 516000 ton/year it is necessary to look for glycerol alternative uses. In this work native microorganisms present in raw glycerol (A and B) were isolated, from two Santander biodiesel plants, and their growth was followed in mineral culture using glycerol USP (24 g/L) as the sole carbon source. Microorganism’s growth was measured by dry weight and absorbance measurements. Glycerol intake by the microorganisms was determined by High Performance Liquid Chromatography-Refraction Index Detector (HPLC-IRD). A consortium consisting of Gram-positive and Gram-negative bacteria and Aspergillus fumigatus fungus was identified in sample A. On the other hand, Bacillus circulans was isolated and identified from sample B. Two of the microorganisms isolated used the glycerol as carbon source.