Biodegradation preference of single, identified bacterium is the basis of bioremediation and microbial enhanced oil recovery (MEOR). However, the impacts of different bacterial species on the biodegradation of polar compounds are unclear. Accordingly, in this study, we performed a 90-day biodegradation simulation of crude oil using Pseudomonas aeruginosa XJ16 and Acinetobacter lwoffii XJ19. Biodegraded oils were characterized by negative-ion electrospray source ionization ESI (-), Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), and gas chromatography-mass spectrometry (GC-MS). We aimed to study the differential biodegradation and mechanism of nitrogen- and oxygen-containing polar compounds using two strains that showed different abilities to produce and utilize polar compounds. P. aeruginosa XJ16 could easily biodegrade n-alkanes and n-alkyl cyclohexanes, whereas A. lwoffii XJ19 was able to utilize complex compounds, such as steranes and hopanes. Compared with P. aeruginosa XJ16, A. lwoffii XJ19 biodegraded more Ni class and produced more intermediate products (N101 and N102 classes). The efficiency of biodegradation of O1 varied with the type of microorganism. Intermediate products, such as alkylphenols, alkylnaphthols naphthols, and phenyl phenols, were produced by P. aeruginosa XJ16, whereas A. lwoffii XJ19 biodegraded these products. After biodegradation, the saturated fatty acids in Pa-90 and Al-90 decreased through the monoterminal, diterminal, and subterminal oxidation of n-alkane. However, naphthenic acids with 1-6 rings produced from terminal oxidation of naphthenes or dearomatization of aromatic hydrocarbons increased. Overall, these findings provided key insights into the biodegradation of polar compounds by different bacterial species.