Novel and miniaturized technique, a dynamic sonication-assisted solvent extraction before the analysis by high performance liquid chromatography-ultraviolet visible (HPLC-UV-Vis) for isolation, identification and quantification of rutin and quercetin metabolites in alcoholic extracts from Bidens pilosa Linné, was developed. The results showed that the flowers have a greater quantity of both analytes than other parts of the plant (e.g., leaves, stem and roots), the results were corroborated by liquid chromatography-mass spectrometry (LC-MS). In addition, the developed extraction technique against traditional methods for metabolites extraction such as solid-liquid extraction, Soxhlet and rotating-disk sorptive extraction was compared. Under optimal conditions of extraction such as 0.3 mL min-1 of solvent flow, ethanol:water (1:1) as solvent type and 0.5 g of sample amount, it was possible to reach 85% of recovery percentage of target analytes and a limit of detection close to 0.1 µg g-1 with a linear range of 50-400 µg g-1 were also obtained. Finally, the antibacterial evaluation of the flower extract of Bidens pilosa Linné, obtained under above optimal conditions against Gram-positive bacteria, was performed. The higher values of the inhibition diameters when using 1000 mg L-1 and significant differences among Staphylococcus aureus, Bacillus cereus, and Listeria monocytogenes were observed. The tests were performed with different microorganisms inoculated from three different absorbance levels (0.05, 0.5 and 0.1 absorbances), at a lower absorbance of these microorganisms in the growth medium used for evaluating the inhibitory effect of the B. pilosa Linné extract, when using this extract at the concentrations of 500 and 1000 mg L-1, statistically higher inhibition diameters were noticed.