Microbes are well known for their harmful as well as beneficial roles in living organisms. It is the harmful effect that needs special attention, due to the occurrence of several microbial-related human diseases. Research communities around the world have worked extensively to isolate and/or synthesize antimicrobial agents to treat such diseases. All these works have resulted in myriad antimicrobial agents to treat human beings, which gave a sense of relief to earlier generations. But the emergence of drug-resistant microbes over a period of time has led the research community to once again look for new and alternative natural antimicrobial agents. Fatty acids, which are ubiquitous in nature, assume significance due to their mild and broad-spectrum antimicrobial properties, easy availability and extremely low toxicity. Research work carried out in the last several decades on antimicrobial fatty acids opens up the opportunities for their application in pharma, food and the cosmetics industry. These safe natural renewable compounds can be used in treating specific infections where the application of conventional antibiotics either failed or is not desirable. The present chapter summarizes antimicrobial activity of fatty acids and their derivatives, target organisms and the proposed mode of actions.