Background: Trinakantamani Pishti (TMP) is a cardio-tonic (Hridya), styptic (Rakta Stambhaka), astringent (Kashaya) formulation frequently used in varieties of bleeding disorders such as bloody diarrhea (Raktatisaara), Raktarsha (bleeding piles), and disorders of excessive menstruation (Atyartava). Still, no published data is available regarding its characterization. Aim: To generate a fingerprint for raw and processed TMP using sophisticated instrumental techniques to assess antimicrobial activity of TMP. Materials and methods: Three samples of TMP were prepared using the standard reference method. Characterization of TMP was carried out by Fourier-transform infrared spectroscopy (FTIR), energy dispersive X-ray analysis (EDEX) with scanning electron microscopy, powder X-ray diffraction (XRD). Antibacterial activity was carried out by the well-diffusion method. Results: Analysis by scanning electron microscope revealed maximum particle size <5 µm and <3 µm in the raw sample and TMP, respectively. Minimum particle size in TMP ranges from 1 to 2 µm and 701 nm. EDEX analysis shows carbon and oxygen as major constituents while Na, Mg, Ca, Si, Fe, and S were present in traces. XRD pattern indicates the amorphous nature of the drug, while FTIR analysis reveals the presence of functional groups such as O-H, CO2, C = O, C-N, N-H. Heavy metals, total microbial count, and microbial limit test were found to be under permissible limits. Anti-microbial study against tested pathogens Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium did not show any effect of TMP. Conclusion: The results of EDEX study showed that Pishti samples have the small particle size i.e., 701nm than the raw i.e., 1-2 µm, which may facillitate absobtion of drug into the body. All heavy metals in the samples were within the permissible limit. Carbon, hydrogen and oxygen are the chief elements of drug which confirms similarity to the Amber, Since the present work is the first published literature on characterization and anti-microbial study on TMP, the outcome can be considered as fingerprint for the drug prepared using the mentioned reference method.