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Richa Singh,1 Laxman Nawale,2 Manisha Arkile,2 Sweety Wadhwani,1 Utkarsha Shedbalkar,1 Snehal Chopade,1 Dhiman Sarkar,2 Balu Ananda Chopade1,3 1Department of Microbiology, Savitribai Phule Pune University, 2Combichem-Bioresource Center, Organic Chemistry Division, National Chemical Laboratory, Pune, 3Dr Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra, India Purpose: Multi- and extensively drug-resistant tuberculosis (TB) is a global threat to human health. It requires immediate action to seek new antitubercular compounds and devise alternate strategies. Nanomaterials, in the present scenario, have opened new avenues in medicine, diagnosis, and therapeutics. In view of this, the current study aims to determine the efficacy of phytogenic metal nanoparticles to inhibit mycobacteria. Methods: Silver (AgNPs), gold (AuNPs), and gold–silver bimetallic (Au–AgNPs) nanoparticles synthesized from medicinal plants, such as Barleria prionitis, Plumbago zeylanica, and Syzygium cumini, were tested against Mycobacterium tuberculosis and M. bovis BCG. In vitro and ex vivo macrophage infection model assays were designed to determine minimum inhibitory concentration (MIC) and half maximal inhibitory concentration of nanoparticles. Microscopic analyses were carried out to demonstrate intracellular uptake of nanoparticles in macrophages. Besides this, biocompatibility, specificity, and selectivity of nanoparticles were also established with respect to human cell lines. Results: Au–AgNPs exhibited highest antitubercular activity, with MIC of |