One novel compound, (R)‐3, 6‐diethoxy‐4‐hydroxycyclohex‐3‐en‐1‐one (1) and thirteen known compounds was isolated from the waste leaves of Nicotiana tabacum. The structures of three compounds (1−3) were confirmed and attributed firstly by the extensive spectroscopic data, including 1D/2D NMR, IR, HR‐ESI‐MS, CD, and ECD spectra. Notably, seven compounds (2, 3, 9, 10, 11, 12, and 13) exhibited better tyrosinase inhibitory activity than the positive control, kojic acid. The binding modes of these compounds revealed that their structure formed strong hydrogen bonds and van der Waals force with the active sites of tyrosinase. These results indicated that waste tobacco leaves are good resources for developing tyrosinase inhibitors.