The excessive population growth and growing energy demand in recent years have led to increased usage of fossil fuels, which contribute to climate change and global warming. This is primarily due to the greenhouse gas emissions from these types of sources. Sustainable biofuels, particularly biodiesel, are preferred as a substitute for fossil fuels to fulfil energy demands. Microorganisms are a vital subject of study in several scientific disciplines. They are everywhere in most environments on Earth, and their use contributes to a minor but notable technological advancement. Microorganisms are being used to produce lignocellulose-derived ethanol for biofuels, a mixture of cellulose, hemicellulose, and lignin found in plant cell walls. Biodiesel from microbes is widely considered due to its advantages, including a short life span, easy access to various environments, and the capacity to remediate several polluted environmental conditions. Presently, the purpose of this chapter is to provide an overview of the different microbial species that are used to produce biofuel.