The rhizosphere is a rich source of actinomycetes which can produce several potential biologically active secondary metabolites. The principal goal for this research is to extract, purify, and characterize the bioactive secondary metabolites produced by three different strains of actinomycetes isolated from the rhizosphere of rosemary, black locust, and olive. The plant growth-promoting effect (PGPE) of the studied strains of actinomycetes onOcimum basilicumL. (basil) and the disease-control effect on necrotic stem lesions of “black leg” caused byFusarium tabacinumon basil were evaluated in silico. The cell-free culture filtrates from the studied actinomycetes isolates were evaluated in vitro for their antimicrobial activity against some common phytopathogens. The secondary metabolites obtained from the cell-free culture filtrates have been chemically characterized using high-resolution electrospray ionization of liquid-chromatography/mass-spectrometric detection (ESI-(HR)Orbitrap-MS). Results of the in silico trial showed that all studied isolates demonstrated PGPE on basil seedlings, improved some eco-physiological characteristics, and reduced the disease incidence ofF. tabacinum. The extracted metabolites from the studied actinomycetes demonstrated antimicrobial activity in a Petri-plates assay. The chemical analysis revealed the presence of 20 different components. This research emphasizes how valuable the examined isolates are for producing bioactive compounds, indicating their putative antimicrobial activity and their potential employment as fungal biocontrol agents. In particular, the obtained results revealed the possibility of green synthesis of some important secondary metabolites, such asN-Acetyl-l-histidinol, Rhizocticin A, and Eponemycin, from actinomycetes. The bioactive metabolites may be successively used to develop novel bio-formulations for both crop protection and/or PGPE.