Aims: To explore the relationship between chemical structures and antimicrobial activities of quaternary ammonium salts (QASs), particularly the impact of hydrophobicity of the salts on the antimicrobial functions. Methods and Results: Four QASs, i.e. 4-aminododecylpyridinium chloride, 4-acetylaminododecylpyridinium chloride, 4-benzoylaminododecylpyridinium bromide and 4-(1-naphthoyl) aminododecylpyridinium bromide were employed in antimicrobial tests against both Gram-negative and Gram-positive bacteria, Escherichia coli and Staphylococcus aureus. These four QASs possess the same long alkyl chain but different hydrophobic substituents at the 4-amino group. Antimicrobial activity of QASs was measured in liquid phases by growing bacterial cultures in the presence and absence of the QAS. The most hydrophobic compound exhibited the strongest antimicrobial activity than other salts. Conclusions: All of the quaternary pyridinium salts exhibited significant antimicrobial activities but in different extents according to their hydrophobicity at the 4-amino position. QASs having larger hydrophobic groups were significantly more effective than that with smaller groups. Significance and Impact of the Study: This research revealed that hydrophobic and aromatic ring structures at 4-amino position on quaternary aminopyridinium ring could improve antimicrobial activity of the salts. The results could assist understanding and development of antimicrobial QASs.