Abstract Background Hospital environmental resources have a significant role in cross-transmission of opportunistic pathogens such as actinomycetes species to the patients. Actinomycetes have a remarkable capability to survive in adverse and harsh conditions of hospital environments; therefore, they are a threat to the health of patients. Due to this issue, we aimed to determine the frequency and diversity of actinomycetes species in hospital soil, water and dust by using a combination of conventional and molecular methods including the phenotypic and biochemical tests for preliminary identification and the PCR amplification of the specific region of the 16S rRNA, hsp65 gene and sequence analyses of 16S rRNA for the genus and species identification. Results A total of 50 (35.2%) actinomycetes isolates from 7 genera were isolated from 142 hospital environmental samples. The three most prevalent species were M. setense 10%, R. erythropolis and M. fortuitum 8% followed by N.cyriacigeorgica and M. gordonae 6%, M. chelonae, M. abscessus, M. lentiflavum, M. mucogenicum, N. asteroides, N. farcinica, R. equi and L. shinushuensis 4% and the single isolates of M. conceptionense, M. septicum, N. rhamnosophilia, N. bravicatena, M. flavescens, M. arupense, M. doricum, M. frederiksbergense, S. heliomycini, S. albus, S. albogriseolus, R. facians, D. maris, G. terae and A. globiformis. Conclusions In conclusion we showed that the hospital environment is a potential reservoir for a broad range of actinomycetes species, due to the remarkable survival capability of these microorganisms in adverse hospital environment, carrying a threat to the health of patients.