|This study investigated the hydrocarbonoclastic microbial community present on weathered crude oil and their ability to degrade weathered oil in seawater obtained from the Gulf St. Vincent (SA, Australia). Examination of the native seawater communities capable of utilizing hydrocarbon as the sole carbon source identified a maximum recovery of just 6.6 × 101 CFU/ml, with these values dramatically increased in the weathered oil, reaching 4.1 × 104 CFU/ml. The weathered oil (dominated by >C30 fractions; 750,000 ± 150,000 mg/l) was subject to an 8 week laboratory-based degradation microcosm study. By day 56, the natural inoculums degraded the soluble hydrocarbons (initial concentrations 3,400 ± 700 mg/l and 1,700 ± 340 mg/l for the control and seawater, respectively) to below detectable levels, and biodegradation of the residual oil reached 62% (254,000 ± 40,000 mg/l) and 66% (285,000 ± 45,000 mg/l) in the control and seawater sources, respectively. In addition, the residual oil gas chromatogram profiles changed with the presence of short and intermediate hydrocarbon chains. 16S rDNA DGGE sequence analysis revealed species affiliated with the genera Roseobacter, Alteromonas, Yeosuana aromativorans, and Pseudomonas, renowned oil-degrading organisms previously thought to be associated with the environment where the oil contaminated rather than also being present in the contaminating oil. This study highlights the importance of microbiological techniques for isolation and characterisation, coupled with molecular techniques for identification, in understanding the role and function of native oil communities.