Free-living amoebae are protists frequently found in water and soils. They feed on other microorganisms, mainly bacteria, and digest them through phagocytosis. It is accepted that these amoebae play an important role in the microbial ecology of these environments. There is a renewed interest for the free-living amoebae since the discovery of pathogenic bacteria that can resist phagocytosis and of giant viruses, underlying that amoebae might play a role in the evolution of other microorganisms, including several human pathogens. Recent advances, using molecular methods, allow to bring together new information about free-living amoebae. This review aims to provide a comprehensive overview of the newly gathered insights into (1) the free-living amoeba diversity, assessed with molecular tools, (2) the gene functions described to decipher the biology of the amoebae and (3) their interactions with other microorganisms in the environment.