A phytochemical investigation on the 5-lipoxygenase (5-LOX) inhibitory methanolic extract of Rudbeckia hirta L. flowers yielded 10 phenolic metabolites, including three phenolic acids, two phenolic acid esters, four flavonol glycosides and a trimethylated flavonol. The structures of the isolated metabolites were determined on the basis of spectroscopic analyses and by comparison with the literature data. Seven of these metabolites were isolated for the first time from the genus Rudbeckia. The in vitro 5-LOX inhibitory, immunomodulatory and antioxidant (oxygen radical absorbance capacity) activities of the isolated compounds were evaluated, and the results provided a new scientific evidence for the ethnopharmacological use of the herb in inflammatory conditions.