The phytochemical investigation of Rudbeckia laciniata L. obtained a new flavonol glycoside (1), together with four flavonol glycosides (2-5) and eight quinic acid derivatives (6-13). The structure was elucidation by means of spectroscopic methods and chemical evidence. The isolated compounds were tested for cytotoxicity against four human tumor cell lines in vitro using the sulforhodamine B bioassay.