Polyunsaturated fatty acids (PUFAs) are essential for human beings to maintain bio functions that would prevent diseases such as depression, neurological diseases, inflammation, autoimmune diseases and cardiovascular disease. Human beings are unable to synthesize essential PUFAs in the body and therefore, they are taken up from different food sources. The major sources of PUFAs are fish, eggs, milk, fruits and seed oil. However, microbes such as microalgae, bacteria and fungi have good capability for the production of PUFAs under controlled conditions. The establishment of a cost‐effective, reliable and eco‐friendly technique for extraction and purification of PUFAs from microbes is a crucial point. This chapter summarizes and describes the various physico‐chemical methods applied to extract and purify PUFAs from microbes.