A suite of three tetraruthenium metallacycles have been obtained from [2+2] self-assemblies between N,N'-Di-(4-pyridyl)-1,4,5,8-naphthalenetetracarbo-xydiimide (4) and one of the three dinuclear arene ruthenium clips, (η(6)-p-iPrC6H4Me)2Ru2(OO∩OO)][OTf]2 (OO∩OO = oxalate 1, 2,5-dioxydo-1,4-benzoquinonato (dobq) 2, 5,8-dihydroxy-1,4-naphthaquinonato (donq) 3; OTf = triflate). All complexes were isolated in good yield (>85 %) as triflate salts and were fully characterized by using (1)H NMR and UV/Vis spectroscopies, and high-resolution electrospray mass spectrometry. A single crystal of the metallarectangle 5 was suitable for X-ray diffraction structural characterization. The biological activities of the metallacycles were determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays, establishing their in vitro anticancer properties. Our results show that for the AGC (gastric cancer) cell lines, the cytotoxicity of (donq)-containing SCC 7 exceeds that of cisplatin, which was used as a control. For HCT15 (colon cancer) cell lines, the cytotoxicity is comparable to both cisplatin and doxorubicin. An in vivo hollow fiber model was used to show growth-inhibitory activity against HCT15 and image-based cytometry experiments indicated that 7 induced apoptosis as the mode of cell death. Complex 7 also showed significant antitumor activity for multidrug-resistant HCT15/CLO2 cell lines, for which doxorubicin was ineffective.