Different types of trehalose containing glycolipids are known to be produced by several microorganisms belonging to the mycolates group, such as Mycobacterium, Rhodococcus, Arthrobacter, Nocardia and Gordonia. Different structures have been elucidated particularly in Rhodococcus genus. Trehalolipids have gained increased interest for their potential applications in a number of fields due to their ability to lower interfacial tension and increase pseudosolubility of hydrophobic compounds. The most widespread application is in bioremediation technologies as such compounds are known to enhance bioavailability of hydrocarbons. In comparison to other microbial glycolipids, trehalolipids have generally showed contrasting results and achievements with both cases of inhibition and enhancement of biodegradation rates. One of the important challenges regarding potential use of trehalose lipids in a variety of applications is the optimisation of their production and downstream processing. In fact, the purification of the target biological compounds by downstream processing can account for over half the production cost in many biotechnology applications. This is especially true in the case of the Rhodococcal glycolipids, which are often bound to cellular envelopes and are usually produced along with other surface active lipids. In this review, we highlight the current knowledge of trehalolipid biosurfactant applications and the latest successful strategies employed to reduce the cost of their production.