Deep-sea sedimentary hydrocarbonoclastic bacteria are still not widely used in the bioremediation field, especially for crude oil spill biodegradation. This study utilized a mixed culture of Raoultella sp., Enterobacter sp., and Pseudomonas sp. isolated from deep-sea sediment to determine the abilities of bacteria to degrade petroleum hydrocarbons while incorporating environmental variations in a microcosm study. The oil biodegradation extent was determined by measuring the remaining oil and grease in the sample vials. The highest percentage of biodegradation was 88.6%, with a constant degradation rate of 0.399 day–1. GC-MS analysis showed that the most degradable compound in the oil samples was paraffin. This study also observed that microbial degradation was optimized within three days of exposure and that degradation ability decreased at 35 °C. The salinity variation effects were insignificant. Based on all analyses, deep-sea sediment bacteria have great potential in oil spill biodegradation in a microcosm scale.