Background: Actinobacteria are widespread and live in a variety of habitats. Today, these bacteria are very important due to the production of various secondary metabolites with different biological activities. The present study aimed to isolate strains of Actinobacteria from different habitats (the Persian Gulf, Gandom Beryan area in the Lut Desert, and some plant roots). The anticancer and antimicrobial activities of secondary metabolites of these isolates were also investigated. Methods: Samples were taken from water of the Persian Gulf, soil of Gandom Beryan area in the Lut Desert, and plant roots. For isolation of Actinobacteria, samples were cultured in ISP2, ISP4, AIA, Gauze, M1, ISP3, and GYP media. Bacterial strains were identified based on the colony and bacterial morphology and confirmed using the specific primers for Actinobacteria. The anticancer and antimicrobial activities of crude metabolite extracts and supernatant of the isolates were evaluated on MCF-7 and Staphylococcus aureus PTCC 112 and Pseudomonas aeruginosa PTCC 1214 strains. Results: The results showed that the supernatants of 7 isolates (ga31, ez, sa, mar2, rz, ga33, and ga5) and the metabolite extracts of 4 strains (ga31, ga5, rz, and ez) had anticancer activity. Overall, ga31 was the best strain with anticancer activity of more than 75%. When evaluating the antimicrobial activity of bacterial secondary metabolites, we found that only two strains (ga31 and ga5) had antimicrobial activity against S. aureus PTCC 1112. Conclusions: In general, strain ga31, which has high anticancer and antimicrobial activities, could be a good candidate for new trials in the pharmaceutical industry.