Traditionally, lipid-producing microorganisms have been obtained via conventional bioprospecting based on isolation and screening techniques, demanding time and effort. Thus, high-throughput sequencing combined with conventional microbiological approaches has emerged as an advanced and rapid strategy for recovering novel oleaginous microorganisms from target environments. This review highlights recent developments in lipid-producing microorganism bioprospecting, following (i) from traditional cultivation techniques to state-of-the-art metagenomics approaches; (ii) related topics on workflow, next-generation sequencing platforms, and knowledge bioinformatics; and (iii) biotechnological potential of the production of docosahexaenoic acid (DHA) by Aurantiochytrium limacinum, arachidonic acid (ARA) by Mortierella alpina and biodiesel by Rhodosporidium toruloides. These three species have been shown to be highly promising and studied in research articles, patents and commercialized products. Trends, innovations and future perspectives of these microorganisms are also addressed. Thus, these microbial lipids allow the development of food, feed and biofuels as alternative solutions to animal and vegetable oils.