Lichen has a great medicinal value and represents the symbiotic relationship between fungi and algae or cyanobacteria. The genus Usnea longissima(Parmeliaceae), is a fruticose lichen with numerous biological activities like antimicrobial, antifungal and inhibitory activities for plant and human pathogens. Taxonomically it is well distinguished by pale greenish to yellowish-green colour and pendulous thallus with dense branches usually emerging from the main stem. Its fungal partner releases an extracellular compound called Usnic acid, a derivative of Dibenzofuran which is a naturally occurring secondary metabolite. In the present work, antimicrobial activities of Usnea longissimi are explored in various extraction solvents. The crude extracts were prepared in methanol, ethanol, ethyl acetate, acetone and different concentrations (2.5,5,10,15,20mg/ml) followed by the determination of its antimicrobial activity against various microbes viz. Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Fusarium oxysporum using agar well diffusion process. The maximum zone of inhibition was observed in the 15 mg/ml methanolic extract for Escherichia coli (34 mm), 10 mg/ml methanolic extract for Staphylococcus aureus (30 mm), 10 mg/ml ethyl acetate for Pseudomonas aeruginosa (16 mm) and 0.5 mg/ml ethanolic extract for Fusariumoxysporium (14 mm). Usnea longissimi have shown significant antibacterial and antifungal activity that encourage us to explore novel antimicrobial components within lichen biodiversity. These lichens further can be used as food supplements to cure various human diseases.