Actinomycetes are gram-positive bacteria that belong to the actinobacterial species. They are a prolific source of secondary metabolites with various biological applications. Thus, this study aimed to culture-based isolation of potent Actinomycetes species from Sof-Umer Cave and in vitro and in vivo evaluation of their potential metabolites against selected test organisms.

Among the total isolates, ten isolates were selected based on their antimicrobial activities. Among them, ethyl acetate crude extract of three isolates (RO13, SD2, R011) showed potential antagonistic activity, ranging from 17 ± 0.78 to 23 ± 0.56 mm of zone of inhibition against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Additionally, two isolates’ (SD2, R011) crude extract exhibited significant inhibition of test organisms in wound and oral infection of the mice models. This was confirmed by wound contraction and progress improvement of the clinical sign observed before treatment. Characterization of their crude extract by FTIR and GC-MS revealed the presence of various functional groups and compounds. Specifically, potent antimicrobial and antioxidant bioactive compounds, such as pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-2-piperidine, phenol, 2-methoxy-4-(1-propenyl) -, and indolizine, were identified via GC-MS analysis. Three of the ten potent isolates (R013, R011, and SD2) were identified based on the 16S rRNA gene sequence, and the R013 isolate belongs to Streptomycetes flavoviridis, whereas SD2 and R011 were identified as Arthrobacter sp. and Actinobacterium kmd_152, respectively.