Hydrophobically modified and unmodified polyvinylamines (PVAm), including a total of five polymers, were tested against both gram-negative (Escherichia coli) and gram-positive (Bacillus subtilis) bacteria for antimicrobial activity. The assessment of PVAm in solution against bacteria is described, and the influence of the charge density and of the hydrophobic modification of the polyelectrolyte is discussed. The antimicrobial activity was found to depend upon the concentration of PVAm and also on the type of bacteria used. The results also indicated that no direct relationship exists between antimicrobial activity and charge density of the different PVAms. It was, however, observed that an alkyl chain length of six or eight alkane units had a substantial effect on the bacteria investigated. The best combined antibacterial activity for the two bacteria tested was achieved for PVAm with a C-6 alkane substituent (PVAm C-6). To evaluate the antimicrobial activity on a solid substrate, PVAm C-6 was further studied after being deposited onto a glass slide and the results show a large reduction in bacterial infection.