A phytochemical investigation of the aerial parts of Mitracarpus hirtus afforded thirteen compounds, including a new naphthoquinone di-glycoside (1), three isopentenyl isoflavones (2−4), four flavonoids (5−8), three iridoid glycosides (9−11), and two coumarins (12 and 13). Their structures were elucidated based on extensive spectroscopic analyses, chemical methods, and the comparison with the literature. Among them, compound 1 possesses a 2-(3-methylnaphthalen-2-yl)acetic acid core with two glucosyl groups, compounds 2−4 are the first three representatives from the Rubiaceae family, and compounds 9−11 and 13 were isolated from Mitracarpus genus for the first time. Additionally, compounds 2−4 displayed antibacterial activities against Helicobacter pylori G27/HP159/JRES00015 (MIC = 4~16μg/mL). To date, wighteone (2) is the most active isoflavone reported against H. pylori.