Biodegradation of marine surface floating crude oil with hydrocarbon degrading bacteria, rhamnolipid biosurfactants, and nutrients was carried out by a large-scale field simulated experiment in this paper. After a 103 day experiment, for n-alkanes, the maximum biodegradation rate reached 71% and the results showed hydrocarbon degrading bacteria, rhamnolipid biosurfactants, and nutrients have a comprehensive effect. It also showed that rhamnolipid biosurfactants could shorten the biodegradation time through an emulsifying function; the nutrients could greatly increase the biodegradation rate by promoting HDB production. For PAHs, the chrysene series had higher weathering resistance. For the same series, the weathering resistance ability is C1- < C2- < C3- < C4-. After 53 days, no comprehensive effect occurred and more biodegradation was found for different n-alkanes in two pools which only had added rhamnolipid biosurfactants or nutrients, respectively. Except for C14, C15 and C16 sesquiterpanes, most of the steranes and terpanes had high antibiodegradability.