AIM AND OBJECTIVE: The aim of this present study is to validate the Anti microbial potentials of a novel siddha metallo-mineral formulation Kaalamega Narayana Chendhooram as mentioned in Athmaraksha Mirtham Ennum Vaithiya Saara Sangeraham against a Gram negative organism Escherichia coli in in-vitro Studies. METHODS: Siddha system of medicine was a fruitful gift offered by the siddhars. It contains plants, metals, minerals and animal products in their medicinal preparations. It involved in treating various acute and chronic diseases. Siddha system of medicine had a history of antimicrobial medicine before the prehistoric era of antibiotics. Due to the lack of proper validations of medicine, it was not accepted by the scientific world. Thus an attempt was made in this paper to screen the efficacy of anti microbial potentials of Kaalamega Narayana Chendhooram as mentioned in Athmaraksha Mirtham Ennum Vaithiya Saara Sangeraham, a novel siddha metallo-mineral formulation against a Gram negative organism Escherichia coli in In-vitro Studies of Muller Hinton Agar Medium with streptomycin as a standard drug and test drug with the different concentration of the drug as 250μg/ml, 500μg/ml, 1000 μg/ml were added and zone of inhibitions were measured in mm. RESULTS: At the end of this research study, that the trail drug of a novel siddha metallo-mineral formulation Kaalamega Narayana Chendhooram as mentioned in Athmaraksha Mirtham Ennum Vaithiya Saara Sangeraham through Muller Hinton Agar Medium with streptomycin as standard drug. The gram negative organism showed the zone of inhibition in following concentration of Escherichia coli 250μg/ml, 500μg/ml, 1000 μg/ml as 15mm,16mm, 19mm .On increasing the concentration of the drug there is a gradual increase in the zone of inhibition of micro organisms. CONCLUSION: The present in-vitro study of a potent siddha metallo-mineral formulation Kaalamega Narayana Chendhooram as mentioned in Athmaraksha Mirtham Ennum Vaithiya Saara Sangeraham through a Muller Hinton Agar Medium with streptomycin as a standard drug was found to be a potent anti-microbial medicine.