The growing demand for alternative energy sources to fossil fuels that can partially or fully replace them has driven a significant increase in research aimed at generating sustainable long-term energy technologies that are environmentally friendly and capable of reversing or halting the environmental impacts caused by conventional fuels. One of the areas of study is the production of bio-oil from microalgae. These microorganisms can produce lipids as free fatty acids (FFAs) or triacylglycerols (TAGs) through their cellular metabolism. These molecules can, in turn, be transformed into biodiesel through various chemical processes. The liquid fuels obtained from microalgae offer significant advantages over other fuels, especially from an environmental perspective, as their production is based on the fixation and consumption of atmospheric CO2 or combustion products, generating oxygen (O2) as a metabolic byproduct. These cultivation systems are cost-effective because they do not require large land areas, and the cultivation media can consist of industrial waste or contaminated water. Additionally, they can utilize natural light as an energy source for photosynthesis. Simultaneously with biodiesel production, other value-added products such as proteins, pigments, and antioxidants can be obtained. All of these characteristics make microalgae a promising source of biofuels. Due to this, this chapter aims to present the most relevant aspects of biodiesel production from microalgae, organized into four main stages of the process: (1) microorganism cultivation and growth, (2) cell disruption, (3) lipid extraction, and (4) chemical or thermochemical conversion of lipids to biodiesel.