In the present study, gold (Au) and silver (Ag) nanoparticles (NPs) were synthesized from aqueous extracts of Opuntia dillenii, characterized by various advanced techniques and investigated for antibacterial and antifungal potential. Phytochemical screening of O. dillenii showed the presence of alkaloids, betacyanin, saponins, tannins, flavonoids, and phlobatannins. The characterizations of the synthesized metal NPs were performed such as UV-visible spectrophotometer, FTIR (Fourier transform infrared) spectrophotometer, SEM (scanning electron microscopy) and EDX (energy-dispersive X-ray). Through the application of such advance techniques, the UV-visible spectrophotometer showed the bands of absorbance for AgNPs and AuNPs at 420 nm and 525 nm range, respectively. The FTIR spectra for both and AgNPs also appeared in the range of 4000-400 cm-1. SEM was performed for the textural and morphological characteristics of the NPs such as shape, size distribution, and surface structure. Elemental analysis was recorded for the synthesis of Au and AgNPs, which confirmed its purity. The O. dillenii extract and their synthesized Au and AgNPs showed a clear zone of inhibition against the E. coli, S. aureus, K. pneumonia, and P. aeruginosa bacteria and T. viride, C. albicans, C. krusei, and A. niger fungal species.