Actinomycetes are filamentous, Gram-positive bacteria of major ecological and biomedical importance due to their exceptional ability to produce diverse bioactive secondary metabolites encoded by numerous biosynthetic gene clusters. In the present study, actinomycetes were isolated from rhizospheric and non-rhizospheric soil samples and systematically characterized using morphological, biochemical, and molecular approaches. The isolates were screened for antimicrobial, antioxidant, and L-glutaminase-producing potential, followed by 16S rRNA gene sequencing for taxonomic identification. Several isolates exhibited strong antibacterial activity against clinically significant pathogens, including Staphylococcus aureus (NCIM 2073) and Escherichia coli (NCTC 9001). Antioxidant potential evaluated using the DPPH radical scavenging assay confirmed the presence of redox-active metabolites. Efficient production of L-glutaminase, an amidohydrolase enzyme was observed. To complement the experimental findings, in silico molecular docking studies were performed to investigate the interaction of selected bioactive compounds with relevant target proteins, providing mechanistic insights into binding affinity and molecular stability. The docking results revealed favourable binding energies and key hydrogen-bond and hydrophobic interactions, supporting the observed biological activities. Overall, the integrated experimental and in silico approach highlights soil-derived actinomycetes as versatile and sustainable bioresources with significant pharmaceutical and biotechnological potential, emphasizing their role in combating antimicrobial resistance and enabling the rational development of novel therapeutic and industrial products.