During biodiesel storage, chemical reactions may occur, producing sludges. The aim of this study was to characterize the chemical and microbial composition of the sludge found in a biodiesel storage tank. The material was collected in a biodiesel production plant. The sludge chemical characterization was performed by Fourier transform infrared (FTIR) spectroscopy, flame atomic absorption spectroscopy (FAAS), gas chromatography (GC), and the microbial investigation used culture-dependent techniques. The deteriogenic potential of the native microbial community was evaluated using the sludge as a microbial inoculum in a 60 day experiment. The microbial growth, biodiesel degradation, pH alterations, and the detection of esters in the aqueous phase were evaluated. The chemical analysis indicated the sludge composition as fatty acids esters and metallic ions; sterols glycosides were not detected. Seven bacteria and five fungi species were obtained from the sludge. The microbial growth analysis indicated that the native community does not have high biodiesel deteriogenic capacity.