Rhamnolipids (RLs) are a group of nontoxic, biodegradable and environment friendly biosurfactants (BSs) produced by microorganisms. Besides being effective surfactants, RLs have been well documented for their antimicrobial, wound healing and skin care activities. This chapter reviews above properties and describes the applications of RLs in agriculture and wound care applications. In agriculture, RLs can be developed as effective biopesticide, for example, in a seed coating to control Phytophthora diseases on soybean plants. The pesticidal activities of RLs on other organisms such as algae, mosquitoes, flies, ants, spiders and grasshoppers support the idea of development of RLs-based algaecide, insecticides and broad-spectrum biopesticide. For wound healing and skin care, RLs have been reported to have certain biological effects beyond the antimicrobial function. A main component in microbe-produced RL mixtures has been demonstrated to have a unique anti-fibrotic property to reduce excessive scarring in wound healing by selective inhibition of myofibroblasts. Further research may lead to the development of RLs-containing ointment and wound dressing products for applications in would healing and skin care.