Lichen-associated endophytic Actinobacteria, particularly Streptomyces species, are recognized for their production of bioactive secondary metabolites with significant pharmaceutical potential. With the escalating prevalence of diseases, Streptomyces species are being investigated for its natural source of antimicrobial compounds for new antibiotics. This study focuses on the bioactive properties of secondary metabolites from lichen-associated endophytic Actinobacteria, focusing on Streptomyces glaucescens NTSB-37 isolated form lichen, Parmotrema perlatum (Huds.) M. Choisy of Kolli hills, Tamil Nadu, India. Among 54 Actinobacterial isolates (NTSB-1 to NTSB-54) collected from various regions in Tamil Nadu, Streptomyces glaucescen NTSB-37 demonstrated notable antibacterial and anti-dermatophytic activities against multidrug-resistant bacteria and dermatophytes, including Trichomonas rubrum and Microsporum canis. The morphological and molecular (16S rRNA gene sequencing) characterization confirmed its identity. Secondary metabolites screening via FTIR and GC-MS revealed bioactive compounds in crude extracts and exhibited increased antibacterial activity with increasing dosage volume and notable MIC values. Partial purification was achieved through TLC and HPTLC fingerprinting. Further investigation shows Streptomyces glaucescens NTSB-37, an endophytic actinobacteria, is a prolific producer of bioactive secondary metabolites with effective antimicrobial agents amid the growing challenge of drug resistance and suggesting biotechnological applications in human health. The findings furthermore emphasize the important of exploring novel ecosystem, particularly lichens with abundant microhabitats within and in hilly regions for developing bioactive compounds with therapeutic potential.