Multilayer films consisting of bovine submaxillary mucin (BSM) and poly(allylamine hydrochloride) (PAH) were prepared on various substrates using layer-by-layer assembly. The effects of both the assembly pH and ionic strength on multilayer characteristics were investigated by assessing film thicknesses (10–80 nm), surface wetting characteristics, and cell repulsion. Also, the dynamic assembly behavior was monitored using quartz crystal microbalance with dissipation monitoring (QCM-D) to further understand the effect of assembly pH on film characteristics. Assembly studies revealed that substantial amounts of BSM adhere to the outermost surface only at low pH conditions. The resulting multilayer films assembled at low pH conditions were found to exhibit hydrophilic and cell repellent behavior. In addition, it was found that batch-to-batch variations of the biopolymer BSM could dramatically alter properties.