Biofuels have been commercialized for decades, predominantly bioethanol and biodiesel, either in pure form or blended to regular fuels gasoline and diesel, respectively. Mostly, they are based on edible feedstock such as corn, sugarcane, rapeseed, and soybean (so-called first-generation (1G) biofuels). The arising competition over arable land with food crops has caused significant debate, as well as a net contribution to climate change, where it was found that sometimes 1G biofuels perform even worse than petroleum-based fuels, due to land use change, fertilizer usage, and process yields, for instance. Compared to other alternative fuels/modes of energy storage like H2 or batteries, biofuels benefit from their high energy densities and compatibility with existing infrastructure. As a result of this, there is a large interest in biofuels for aviation, so-called sustainable aviation fuels (SAF), and for marine applications. Biofuel research has traditionally targeted lignocellulosic feedstock, which exists in abundance. Due to the recalcitrance of these biopolymers, cost-effective 2G (second-generation) biofuels are still only on the verge of commercialization. The critical step to break up the biomass can be either a thermochemical or a biochemical process, using enzymes. Going further, 3G (third-generation) biofuels have been envisioned, where autotrophic microorganisms are deployed. For instance, since algae can form up to more than an order of magnitude more biomass per area than terrestrial biomass, they hold great promise for future biofuel production on marginal land or in the ocean. Also, bacteria that feed on gaseous compounds, such as synthesis gas, carbon oxides, or methane, are highly attractive candidates for large-scale, low-environmental impact and cost-effective biofuel production, and gas fermentation has recently been successfully commercialized. A further expansion are 4G biofuels that are carbon-negative. In this chapter, 2G and particularly 3G biofuel concepts, where bacteria and algae are used to obtain biofuels, are discussed. Standard industrial processes, like ethanol fermentation from starch/sugar through microorganisms for regular 1G biofuels and transesterification of various oils to fatty acid methyl esters (FAME, biodiesel), are not covered here. Alternative biofuels from bacteria and algae, such as biomethanol or biohydrogen, are also addressed.