We synthesized gemini quaternary ammonium compounds (gemini QACs) having two thiazolium moieties in a molecule, 5,5′-[2,2′-(α,ω-polymethylnedicarbonyldioxy)diethyl]bis(3-alkyl-4-methylthiazolium iodide) (5DEBT-m,n), on which the carbon number of the methylene chain linking the two thiazoles (m) is 2, 6 or 8 and that of the alkyl group (n) is 8, 10, 12, 14 or 16. 5,5′-[2,2′-(p-Phenylenedicarbonyldioxy)diethyl]bis(3-alkyl-4-methylthiazolium bromide) (5DEBT-P,n) was then synthesized, which is composed of a p-phenylene as the methylene spacer. For five gemini QAC series, in addition to the previously described 5DEBT-4,n to the four new compound series, their antimicrobial activities were determined. 5DEBT-m,10 and -P,10 exhibited a wide and strong bacteriostatic activity against gram-negative and -positive bacteria, fungi and then yeast in comparison with N-tetradecyl-5-(2-hydroxyethyl)-4-methylthiazolium iodide as a mono-QAC. The bactericidal activity of the 5DEBT series against Escherichia coli IFO 12713 and Staphylococcus aureus IFO 12732 was investigated on the basis of the effects of their alkyl chain length and their molecular hydrophobicity. It was found that the effect of theses factors on their activity significantly changes by the difference between the gram-negative and -positive bacteria. Although against the gram-negative bacterium, the change in the activity due to extension of the alkyl group for each compound affected the kind of methylene spacer, against the gram-positive bacterium, it was almost equal in spite of the methylene spacer. This result could be responsible for the bactericidal mechanism of the gemini QACs being influenced by the diversity of the steric structure participating in the methylene chain length and by the bacterium cell surface hydrophobicity.