In the current study, the actinomycetes associated with the red sea‐derived soft coral Sarcophyton glaucum were investigated in terms of biological and chemical diversity. Four different media, M1, ISP2, Marine Agar (MA), and Actinomycete isolation agar (AIA) were used for the isolation of three strains of actinomycetes that were identified as Streptomyces sp. UR 25, Micromonospora sp. UR32 and Saccharomonospora sp. UR 19. LC‐HRMS analysis was used to investigate the chemical diversity of the isolated actinobacteria. The LC‐HRMS data were statistically processed using MetaboAnalyst 5.0 viz to differentiate the extract groups and determine the optimal growth culturing conditions. Multivariate data statistical analysis revealed that the Micromonospora sp. extract cultured on (MA) medium is the most distinctive extract in terms of chemical composition. While, the Streptomyces sp. UR 25 extracts are differ significantly from Micromonospora sp. UR32 and Saccharomonospora sp. UR 19. Biological investigation using in vitro cytotoxic assay for actinobacteria extracts revealed the prominent potentiality of the Streptomyces sp. UR 25 cultured on oligotrophic medium against human hepatoma (HepG2), human breast adenocarcinoma (MCF‐7) and human colon adenocarcinoma (CACO2) cell lines (IC 50 =3.3, 4.2 and 6.8 μg/mL, respectively). SwissTarget Prediction speculated that among the identified compounds, 16‐deethyl, indanomycin ( 8 ) could have reasonable affinity on HDM2 active site. In this respect, molecular docking study was performed for compound ( 8) to reveal a substantial affinity on HDM2 active site. In addition, molecular dynamics simulations were carried out at 200 ns for the most active compound ( 8) compared to the co‐crystallized inhibitor DIZ giving deeper information regarding their thermodynamic and dynamic properties as well.