Choline kinase (ChoK) is the first enzyme in the CDP-choline pathway that synthesizes phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. Human ChoK has three isoforms: ChoKα1, α2, and β. Specific inhibition of ChoKα has been reported to selectively kill tumor cells. In this study, ten new symmetrical bis-pyridinium and bis-quinolinium derivatives were synthesized and tested for their ability to inhibit human ChoKα2. These compounds have electron-releasing groups at position 4 of the pyridinium or quinolinium rings. 1,1′-[(Butane-1,3-diylbis(benzene-1,4-diylmethylene)]bis[4-(4-bromo-N-methylanilino)pyridinium)] dibromide and 1,1′-(biphenyl-3,3′-diylmethylene)bis[7-chloro-4-(perhydroazepine-1-yl)quinolinium] dibromide were identified as highly potent ChoK inhibitors with IC50 values of 80 nM. Kinetic enzymatic assays indicated a mixed and predominantly competitive mechanism of inhibition for these compounds, which exhibited strong antiproliferative activity (EC50 1 μM) against the human breast cancer SKBR3 cell line.