Brewery spent grain (BSG) has previously been exploited in bioremediation. However, detailed knowledge of the associated bacterial community dynamics and changes in relevant metabolites and genes over time is limited. This study investigated the bioremediation of diesel contaminated soil amended with BSG. We observed complete degradation of three total petroleum hydrocarbon (TPH C10–C28) fractions in amended treatments as compared to one fraction in the unamended, natural attenuation treatments. The biodegradation rate constant (k) was higher in amended treatments (0.1021k) than in unamended (0.059k), and bacterial colony forming units increased significantly in amended treatments. The degradation compounds observed fitted into the elucidated diesel degradation pathways and quantitative PCR results showed that the gene copy numbers of all three associated degradation genes,alkB,catAandxylE,were significantly higher in amended treatments. High-throughput sequencing of 16S rRNA gene amplicons showed that amendment with BSG enriched autochthonous hydrocarbon degraders. Also, community shifts of the generaAcinetobacterandPseudomonascorrelated with the abundance of catabolic genes and degradation compounds observed. This study showed that these two genera are present in BSG and thus may be associated with the enhanced biodegradation observed in amended treatments. The results suggest that the combined evaluation of TPH, microbiological, metabolite and genetic analysis provides a useful holistic approach to assessing bioremediation.