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The objectives of this study were (1) to investigate the effect of extracts from some plants in the families Nelumbonaceae and Nymphaeaceae on phosphodiesterase 5 (PDE5) and arginase, which have been used in erectile dysfunction treatment, and (2) to isolate and identify the compounds responsible for such activities. The characterization and quantitative analysis of flavonoid constituents in the active extracts were performed by HPLC. Thirty-seven ethanolic extracts from different parts of plants in the genus <i>Nymphaea</i> and <i>Victoria</i> of Nymphaeaceae and genus <i>Nelumbo</i> of Nelumbonaceae were screened for PDE5 and arginase inhibitory activities. The ethanolic extracts of the receptacles and pollens of <i>Nelumbo nucifera</i> Gaertn., petals of <i>Nymphaea cyanea</i> Roxb. ex G.Don, <i>Nymphaea stellata</i> Willd., and <i>Victoria amazonica</i> (Poepp.) Sowerby and the petals and receptacles of <i>Nymphaea pubescens</i> Willd. showed IC₅₀ values on PDE5 of less than 25 μg/mL while none of the extracts showed effects on arginase. The most active extract, <i>N. pubescens</i> petal extract, was fractionated to isolate and identify the PDE5 inhibitors. The results showed that six flavonoid constituents including quercetin 3’-O-<i>β</i>-xylopyranoside (<b>1</b>), quercetin 3-methyl ether 3’-O-<i>β</i>-xylopyranoside (<b>2</b>), quercetin (<b>3</b>), 3-<i>O</i>-methylquercetin (<b>4</b>), kaempferol (<b>5</b>) and 3-<i>O</i>-methylkaempferol (<b>6</b>) inhibited PDE5 with IC₅₀ values at the micromolar level.