Aims The aim of this study was to elucidate the chemical properties and applications of trehalose lipids produced by Rhodococcus qingshengii strain FF and optimize its production yield. Methods and Results Strain FF was identified as R. qingshengii. It was observed to produce biosurfactants in the presence of n-hexadecane. The biosurfactants were identified as the mixture of trehalose triesters and trehalose tetraesters, mainly consisting of TrehC(12)C(3)C(6)C(12):10, TrehC(11)C(8)C(6):6, TrehC(11)C(6)C(4):5 and TrehC(6)C(4)C(6):5 based on the analysis of thin layer chromatography, Fourier transform infrared and flight tandem mass spectrometry. The best carbon source and nitrogen source for producing trehalose lipids was the mixture of n-hexadecane and oleic acid (m : m = 1 : 1) and the organic nitrogen, urea. Under this condition, the production of trehalose lipids could reach 7 center dot 97 g l(-1). The crude trehalose lipids showed extremely high surface-active properties and were proven to promote the degradation of naphthalene. Conclusions The trehalose lipids produced by R. qingshengii strain FF exhibited high surfactant activity under various conditions and were proven to promote the degradation of naphthalene. Significance and Impact of the Study Rhodococcus qingshengii strain FF is a potential candidate for bioremediation. The trehalose lipids might be used as unique biosurfactants in cosmetic industries, biological formulations and other applications.