Actinobacteria of the order Actinomycetales are ubiquitous in the world. They are found in many habitats ranging from terrigenous, marine, aquatic, aerial and extreme environments as well as in association with macro- and higher organisms. Since the discovery of first antibiotics from the members of this order in the 1940s, significant attention was directed towards their detection and isolation due to their metabolic diversity. However, most of the earlier studies were done using dilution plate techniques that limited a direct means of assessment of true actinobacterial diversity. Most studies then utilized random approaches, involving large-scale, indiscriminate sampling of the environment and the subsequent screening of the isolates for detection of bioactive compounds for pharmaceutical and industrial use. These early approaches remained inadequate in terms of generation of sound understanding related to existence and the functional diversity of the actinofloral layers of natural environments. However, current molecular advances such metagenomics are now aiding towards generation of in-depth understanding on actinobacteria in natural environments. This chapter will thus overview the impact of current molecular advances on the generation of in-depth understanding on the true or transitory occurrence, diversity and eco-functional roles of Actinomycetales in diverse habitats.