Background. Rubia cordifolia has been used to treat various diseases and exhibits antimicrobial, anti-inflammatory, analgesic and antidiarrheal activities. The plant has not been investigated for its anti-mycobacterial activity against virulent tuberculosis strains as well as cytotoxic and anti-oxidant activities in mononuclear cells and neutrophils. Methods. Ethanolic and dichloromethane leaf extract fractions of Rubia cordifolia (0.2-125µg/ml) were screened for anti-mycobacterial activity using a fluorescent microplate assay. The anti-oxidant activity of the fractions was tested using the radical scavenging assay, while their cytotoxicity to lymphocytes was measured using the WST-8 assay. Reactive oxygen species production by neutrophils exposed to fractions was tested using Cell Meter™ Fluorimetric ROS Assay kit. Results. The ethanolic leaf fractions exhibited overall superior anti-mycobacterial activity than the dichloromethane group and were further screened for anti-oxidant and cytotoxic activities. Ethanolic sub-fractions K2F-3.2, K2F-3.3 and K2F-3.4 showed significant antioxidant activities at concentration of 50 µg/ml to 200 µg/ml and higher in both the cell free-based radical scavenging activity and total reactive oxygen species production assays. For lymphocytes, the test agents showed anti-proliferative activity at 25 µg/ml to 200 µg/ml for sub-fraction K2F-3.3B and 50 µg/ml to 200 µg/ml for agents K2F-3.2 and K2F-3.4. Conclusion. The current study is the first to record the in vitro anti-mycobacterial, anti-oxidant and cytotoxic activities of Rubia cordifolia plant leaf extract sub-fractions using the given experimental setups and further research activities to identify the bioactive components are to be pursued.