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DNA multilayer films are promising candidates for a plethora of applications, including sensing, diagnostics, and drug/gene delivery. Fabricated solely from DNA, the use of salt in forming DNA multilayers is crucial in promoting and maintaining hybridization of complementary base pairs by minimizing the repulsive forces between the oligonucleotides and preventing disassembly of the layers once formed. Herein, we examine the role of salt on the assembly of DNA films assembled from oligonucleotides composed of two homopolymeric diblocks (polyA(n)G(n) and polyT(n)C(n)) in salt concentrations ranging from 0.1 to 2 M. Using quartz crystal microgravimetry (QCM) and flow cytometry, we show that films assembled at high salt concentrations (2 M salt) exhibit a different morphology and are denser than those assembled from lower (1 M salt) salt solutions. Formation of the T x A*T triplex in solution and within the DNA film was also studied using circular dichroism (CD) and QCM, respectively. DNA films assembled using oligonucleotides of various lengths (20- to 60-mer) at high salt concentration (2 M salt) showed no significant influence on the film growth. This work shows that salt plays an important role in the assembly and final morphology of DNA multilayer films, hence enabling films with different properties to be tailored.