The leaf extracts of dried and fresh Green and Black Tulsi were prepared in aqueous condition, and employed for the synthesis of AgNPs, using silver nitrate solution. The synthesis was confirmed by color change and was characterized using UV–Vis spectroscopy (for nanoparticle formation), FTIR (for analysis of functional group involved in reducing and stabilizing of nanoparticles) and FESEM (morphological, size analysis). Antibacterial activity was determined against multidrug resistant bacteria (ESKAPE pathogens) by Minimum Inhibitory Concentration (MIC) assay while antifungal activity was tested against Aspergillus niger through agar well diffusion. Results The UV-Vis absorption peak around 400 nm observed for the prepared AgNPs indicates their formation. FTIR investigation revealed the existence of the functional groups of OH and carbonyl, which are mainly responsible for the reducing of Ag+ ions. Non-uniform morphology with particle size between 100–200 nm was observed under FESEM images. The AgNPs displayed potent antibacterial activity on multi drug resistant bacteria due to their combined actions with the silver and the Tulsi phytochemicals. Antifungal assay the antifungal tests indicated zone of inhibition against A.niger. Conclusion Green synthesized AgNPs employing Tulsi extracts is a cost effective, eco-friendly and scalable method producing the AgNPs with enhanced antimicrobial activity.