The major study of this work was to evaluate the chemical composition and the inhibitory components of Parmotrema perlatum against the clinically isolated specimens of Staphylococcus, Streptococcus, Escherichia, Pseudomonas and Candida.. The GC-FID-MS by using a Trace GC Ultra apparatus MS DSQ II detectors and FID-MS splitter under the operating conditions of apolar capillary column was employed to determine the chemical composition of Parmotrema perlatum essential oil. Amino acid (38.1%) and cymene (29.1%) were the most adequate constituents in this oil. The antibacterial activity of Parmotrema perlatum oil was tested against standard strains of bacteria and clinical strains isolated from patients with infections of the oral cavity, abdominal cavity, respiratory, genitourinary tracts, skin, and from the hospital environment. Kirby-Bauer disc diffusion technique was employed to determine the susceptibility of the organism towards the Parmotrema perlatum essential oil exhibited very strong antimicrobial activity to inhibit the growth of the clinical specimens. Application of natural antimicrobial based essential oil from Parmotrema perlatum can be potential source in the treatment & prevention of different types of infections as an alternative to the chemical - based antibiotics based on the investigation.