A lichen is a composite organism formed of algae or cyanobacteria that live in a mutually advantageous symbiotic relationship with the filaments (hyphae) of fungus. Three lichen samples were obtained from diverse sites at a terrestrial habitat located in Coimbatore and coastal habitats located in Kanyakumari and Nagapattinam districts of Tamil Nadu. Amplification and sequencing of 16S rRNA V3–V4 regions were used for metagenomic study. Aside from the Next-Generation Sequencing data (NGS), distinct types of lichen microbiome profiles were clearly revealed. The bacterial diversity in the lichen genera of Roccella montagnei growing in coastal and terrestrial environments was further investigated using common and unique operational taxonomic units (OTUs) and the QIIME pipeline (1.9.1). Using similarity clustering, the heat map analysis depicts the abundance information of chosen OTUs as well as the similarity and difference between OTUs and lichen samples. Using multiple methods, the alpha and beta diversity analysis revealed that there were differences in all of the samples. However, UPGMA tree inference of comparable bacterial community in coastal habitat lichen samples compared to terrestrial habitat validates their evolutionary lineage. As a result, the bacterial population associated with corticolous lichen is dependent on geographic locations, growth substrate, and climatic circumstances of similar lichen genera produced in different habitats and tree substrates. Keywords: Lichen · Roccella montagnei · Microbiome · 16S rRNA V3–V4 regions · OTUs · QIIME.