In this work, the aqueous leaf extracts of three Ophiorrhiza genus species, namely Ophiorrhiza mungos (Om), Ophiorrhiza harrisiana (Oh) and Ophiorrhiza rugosa (Or), have been used as the reducing and capping agents to control the size of AgNPs, Om-AgNPs, Oh-AgNPs and Or-AgNPs, respectively and found to be an effective antimicrobial agent against a wide range of bacteria and fungi. The biosynthesized AgNPs were studied by UV–Visible spectrophotometer, powder X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive X-ray, transmission electron microscopy (TEM) and Fourier transform infrared spectrometer (FTIR). The average particle sizes of Om-AgNPs, Oh-AgNPs and Or-AgNPs were measured as 17 nm, 22 nm and 26 nm, respectively, and observed to be spherical and face-centered cubic crystals. The antibacterial test of synthesized AgNPs was performed against Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Vibrio cholerae where the maximum antibacterial activity was observed by reducing the nano-size and increasing the silver content of AgNPs. The antifungal effect of these three types of AgNPs on Penicillium notatum and Aspergillus niger was also evaluated and their growth with AgNPs concentrations of 450 μg/mL was inhibited up to 80–90% and 55–70%, respectively. The size-control synthesis of AgNPs using the Ophiorrhiza genus species is presented here for the first time where the synthesized AgNPs showed higher stability and antimicrobial activities. Therefore, this study might lead to synthesize AgNPs with different morphologies using plant extracts of the same genus but from different species and provide strong encouragement for future applications in treating infectious diseases.