The global threat of tuberculosis demands the search for alternative antimycobacterial drugs. The present study examined roots and petioles from Rheum rhaponticum for antimycobacterial activity. Crude methanol extracts and eight phenolic compounds isolated by preparative column chromatography were tested against Mycobacterium tuberculosis H37Ra and M. bovis using the broth dilution method. The extract from roots and its components, such as rhaponticin, deoxyrhaponticin, resveratrol, barbaloin, aloe-emodin, and chrysophanol were found to have antimycobacterial activity against both microorganisms. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration of all the investigated samples ranged from 32 to 512 microg/mL. The anthracene derivatives were the most active; their MICs were 32, 64, and 64 microg/mL (M. tuberculosis H37Ra) and 128, 64, and 64 microg/mL (M. bovis), respectively. The microorganisms were resistant to stimulation with extract from petioles, as were quercetin and rutin. The results showed that anthracene and stilbene derivatives play a prominent role in the antimycobacterial properties of R. rhaponticum.