The rise in production of Carbon out of pollution, medical sciences have started re-using the Carbon for treating various diseases because of it’s versatile character as a molecule. Because of the use of Carbon as a carrier molecule, the characterization of the Carbon Doped Cephalexin nanoparticles is also conducted in this work. The appearance of the gel is ash-black in colour depending on the concentration of Carbon used in formulation. The aim of the present work is to formulate Carbon doped Cephalexin Nanoparticles as topical gel for testing its anti-microbial activity and also to check it’s mechanism of action with the help of bio-film formation. In this article, the anti-microbial activity of the Carbon doped Cephalexin nanoparticle gel is tested against organisms like Protease, E.coli, and Staphylococcus sp. and Bacillus substilis as well as it’s mechanism of action is tested using anti-biofilm production. The phase 0 of the clinical trials is conducted in this work in order to understand its anti-microbial characteristics. After formulating various batches of the Carbon doped Cephalexin topical gel based on varying concentration of Carbon, CC5 has been able to show the highest property of gel characteristic out of all the batches. Followed by several evaluation tests being conducted including the microbial assay, it was found that CC2 and CC5 showed the highest zone of inhibition, that is, highest anti-microbial activity out of all batches. The formulations has hence proven to show their respective anti-microbial activity against broad-spectrum of microbes with their respective mechanism of action being studied using the bio-film preparation and its ability to rupture the bio-film produced by the microbes.