| Summary: | Horchata, a herbal infusion drink from Ecuador containing a mixture of medicinal plants, has been reported to exhibit anti-inflammatory, analgesic, diuretic, and antioxidant activity. The antibacterial activity of each of the plants contained in the horchata mixture has not been fully evaluated. Thus, in this study, we analysed the antibacterial activity of 21 plants used in horchata, collected from the Ecuadorian Andes region, against bacterial strains of clinical importance. The methanolic extract of <i>Cinnamomum</i> sp. showed minimal inhibitory concentration (MIC) values of 250 µg/mL against <i>Staphylococcus aureus</i> ATCC25923 and Methicillin-resistant <i>S. aureus</i> (MRSA), while <i>Pelargonium odoratissimum</i> exhibited a MIC value of 500 µg/mL towards <i>S. aureus</i> ATCC25923. The high-performance liquid chromatography-diode array detector-tandem mass spectrometry (HPLC-DAD-MS/MS) analyses identified in <i>Cinnamomum</i> sp. epicatechin tannins, cinnamaldehyde, and prehelminthosporol molecules, whereas in <i>P. odoratissimum</i>, gallocatechin and epigallocatechin tannins, some flavonoids, and gallic acid and derivatives were identified. Finally, <i>Cinnamomum</i> sp. and <i>P. odoratissimum</i> showed partial inhibition of biofilm formation of <i>S. aureus</i> ATCC25923 and MRSA. Overall, our findings revealed which of the plants used in horchata are responsible for the antibacterial activity attributed to this herbal drink and exhibit the potential for <i>Cinnamomum</i> sp. and <i>P. odoratissimum</i> secondary metabolites to be explored as scaffolds in drug development.
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