In this work, we designed contact-killing ionically cross-linked polymeric thin films using Layer-by-Layer (LbL) technology. A polycation, N,N-dodecyl,methyl-polyethylenimine, with microbicidal activity was layered with a polyanion, such as poly(acrylic acid), to create LbL films highly effective against both airborne and waterborne Escherichia coli and Staphylococcus aureus (Gram negative and positive bacteria, respectively), as well as influenza A/WSN (H1N1) virus. The dependence of the microbicidal activity on the pH during and post-assembly of LbL film formation, the nature of the polycation and polyanion, the number of layers in the LbL film, and other experimental variables was investigated quantitatively.