The cultivable actinomycetes from tropical limestone caverns at Khao No-Khao Kaeo karst, Thailand, were investigated. In total, 276 actinomycetes were isolated from 10 soil samples using different selective isolation procedures. The predominant actinomycetes from all samples were members of the genus Streptomyces (94.6%) as they contained LL-diaminopimelic acid (A 2pm) in their whole cell hydrolysates. The remaining 15 isolates (5.4%), rich in meso-A 2pm in whole cell hydrolysates were characterized by 16S rRNA gene sequencing. They were shown to represent strains from 8 different genera namely Actinomadura, Actinoplanes, Gordonia, Microbispora, Micromonospora, Nocardia, Nonomuraea and Saccharopolyspora. A good agreement was found between the results based on morphology, presence of meso-A 2pm and 16S rRNA gene phylogeny. Microwave treatment of soil samples was found to promote the isolation of rare actinomycetes. Successful isolation of members of the genus Actinomadura and Saccharopolyspora from caves was reported for the first time. This study confirms significant diversity of cultivable actinomycetes from neglected habitats such as limestone caves in Thailand.