© 2020, Turkish Chemical Society. All rights reserved.Actinobacteria bacterial group has one of the most populous population in microorganisms that extends very different and extensive habitats on earth especially the main character of the natural soil habitats. Since old times, bioactive metabolites of soil microorganisms have been studied and the results have provided that metabolites of these microorganisms have significant benefits to science, medicine, agriculture, and the pharmaceutical industry. In this study, isolation of Actinobacteria strains from forest soils, identification of morphological and molecular features, extraction of the bioactive metabolite of isolates and determination the antimicrobial, antibiofilm, and cytotoxic activities of bioactive extract were tested. The microbiological isolation methods for collected forest soil samples were used and after the determination of their morphological and molecular features, isolates were defined as Brevibacterium spp. that is a member of Actinobacteria. The antibiotic resistance of the isolates was determined by different methods and different concentrations of standard antibiotics. The chromium tolerance of isolates was also determined. The bioactive metabolites of isolates were produced in a modified medium and extracted. The antimicrobial, antibiofilm, cytotoxic activities of bioactive metabolites were determined against standard microorganisms and Artemia salina larvae were used as a test organism for cytotoxic tests. In the present study, results provided information about Actinobacteria that were isolated from forest soils. Isolates have antibiotic resistance and chromium tolerance abilities. Moreover, it has been shown that the Actinobacteria group is the largest bioactive metabolite producing group in terms of both antibacterial and antifungal activity and also contains a wide range of other compounds such as antibiofilm and cytotoxic compounds. The antimicrobial (MIC) concentrations of bioactive metabolites were detected 2500 μg/mL for standard bacteria cultures and 1250 μg/mL for yeasts. The antibiofilm (MBEC) value was determined at 1250 μg/mL. The 2500 μg/mL concentration of the extract was found to be the effective cytotoxic value. The results provide Brevibacterium spp. isolates have industrial and pharmaceutical potential and more detailed pharmaceutical researches are planned.