Introduction: Antimicrobial agents are essentially important in reducing the global burden of infectious diseases. Neelanjana mai is a classical siddha herbo-mineral combination which is indicated for eye disease in the sastric siddha text ‘Siddha Vaidhya Thirattu”.Methods: In this study, we evaluated the antimicrobial potency of this drug on 3 bacterial species like Staphylococcus aureus,Pseudomonas aeruginosa, Chlamydia trachomatis with Ciprofloxacin and vancomycine as standard controls ;and 3 fungal species like Candida albicans Aspergillus niger, Fusarium oxysporum with Fluconazole as standard controls, using Agar disk- diffusion method. Results: The observed results showed potential antibacterial potency of Neelanjana Mai against the bacterial strains tested with ZOI 17±2 against P.aeruginosa and 14.67±1.53 and 15±1.55 against S.aureus and C.trachomatis respectively. Also, anti-fungal activity results revealed that against the 3 fungal strains. C.albicans, A.niger and F.oxysporum showed anti-fungal effect with ZOI 12.67±1.15, 18.67±1.53 and16.67±2.08 respectively. Conclusion: In summary, overall the observed results concluded that Neelanjana mai extract was effective on P.aeruginosa and A.niger with highest ZOI and confirmed the anti-microbial effect