Background: Screening of antibacterial compounds from natural sources for social benefit is emphasized in recent years with the view that pathogens have developed resistance against antibiotics. Objectives: The present investigation has been carried out to screen sub-aerial cyanobacteria thriving in an extreme environment as they are supposed to be a potential source of natural bioactive compounds. Methods: Scytonema ocellatum isolated from building facades were used for antibacterial activity against certain reference human pathogenic bacteria after subjected to TLC purification twice by altering the solvents. Results: The acetone extract of S. ocellatum showed highest and moderate inhibition zone against Escherichia coli and Pseudomonas aeruginosa respectively. Chloroform extracts also exhibited significant zone against Escherichia coli; no activity was observed against Bacillus subtilis and Staphylococcus aureus. The UV illuminated bands with potential effect were subjected to GC and FTIR analysis. GC analysis showed 98% and 95.6% purity in each solvent extracts and FTIR spectra showed stretching bands containing various biologically active functional groups like alcohol, phenol, alkanes, carboxylic acids groups, amines etc respectively revealing antibiosis. Conclusion: The above screening is the first record of antibacterial activity enhancing the importance of sub-aerial cyanobacteria that possess a scope for producing biologically active compounds, pointing towards brilliant candidates for Pharmaceutical application.