This work was aimed at to extract a novel antimicrobial compound, especially against certain human pathogens, from a sub-aerial cyanobacterium Fischerella sp., isolated from the building facades of Bhubaneswar, Odisha (India). The antimicrobial activity was determined in different solvents and aqueous extracts using agar well diffusion method which were purified using column chromatography and TLC (twice), followed by direct bioautography, MIC, cytotoxicity and was characterized using UV-Vis, FTIR and LC-MS spectroscopic methods. The lowest MIC value (0.312 and 0.625 mg mL-1) was depicted by the acetone extracted single compound against Escherichia coli and Candida albicans. The bioautography and spectroscopic analysis revealed the compound to be an alkaloid which was identified as pentaphenyl ferrocene carboxamide and found to be non-toxic against Catla thymus macrophage and Mus musculus up to 72 h. The cyanobacterium was identified as Fischerella sp. (accession No. MN593556) through 16S rRNA technique. The compound has potential for use as a therapeutic drug. © 2021, ACS Publisher. All rights reserved.