Actinobacteria are a group of Gram-positive microorganisms with a high G+C content in their DNA and belong to the phylum Actinobacteria, one of the largest phyla within bacteria. Some of these actinobacteria have an endophytic lifestyle which occurs abundantly in most plants. The abundance and diversity of endophytic actinobacterial colonisation depend on plant species, type of soils and other associated environmental conditions. Streptomyces spp. were reported as the most predominant species, and Microbispora, Micromonospora, Nocardioides, Nocardia and Streptosporangium are other common genera of endophytic actinobacteria isolated from a diverse range of plant species, including those found in estuarine/mangrove ecosystems and algae and seaweeds of marine ecosystems. Over the years, isolation media have been devised and numerous methods have been standardised for the isolation, identification and characterisation of these endophytic actinobacteria. Recent advances in molecular tools have revealed the 'not yet cultured' diversity within this group. Therefore, a combination of both culture-based and molecular techniques is essential to describe the diversity and ecology of endophytic actinobacteria. The quest for actinobacteria and their metabolic capabilities is ongoing, as they represent the largest ecological resource for secondary metabolites (plant hormones, antibiotics and other bioactive compounds), with potential biotechnological applications in agriculture, industry and medicine.