Biodiesel production, in which glycerol is produced as a by-product, has increased significantly and now vast amounts of glycerol have been considered as a waste product. Biotechnological conversion of glycerol into a valuable bioplastic raw material, namely, 1,3-propanediol (1,3-PDO) seems to be very promising. In this study, locally isolated Klebsiella pneumoniae and Clostridium beijerinckii NRRL B593 were comparatively studied for their 1,3-PDO production potential with the addition of different concentrations of glucose together with glycerol. Addition of a co-substrate enhanced the molar yields of 1,3-PDO by 11% only for K. pneumoniae with an addition of 1 g/L of glucose.