Ticks are hematophagous arthropods which transmit bacterial, viral, and protozoan pathogens. The interaction of a tick's innate immune response with transmitted pathogens is directly linked to its vector competence. The American dog tick, Dermacentor variabilis vectors the etiologic agents of Rocky Mountain spotted fever (Rickettsia rickettsii) and tularemia (Francisella tularensis). Previous studies demonstrated that transcript abundance of two defensins from D. variabilis increases in response to the model Spotted Fever Group organism Rickettsia montanensis. Based on this observation we hypothesized that these defensins function against tick-borne pathogens to limit bacterial load, enabling D. variabilis to serve as vector and reservoir. This study demonstrates that these phylogenetically distinct peptides are also distinct in their antimicrobial mechanism of action and specificity for transmitted bacterial species. Defensin-2 associated with R. montanensis bacteria in vitro and in vivo, inducing cytoplasmic leakage and reducing host burden. Meanwhile, defensin-1 treatment did not reduce host burden of R. montanensis but reduced CFUs of the model organism Francisella novicida in vitro. This reduction in viability correlated with hyperpolarization, potassium efflux, and ATP reduction in the bacteria, factors important for maintaining homeostasis. Additionally, lipid A mutants of F. novicida demonstrated altered susceptibility to and activation of defensin-1 implicating the importance of bacterial membrane components in a successful tick immune response. Finally, this work developed a method of using translation occluding Peptide Nucleic Acids (PNA) to inhibit Rickettsial protein expression. Targeting OmpB and RickA, factors established as important to infection, reduced expression and bacterial burden of host cells. This strategy provides a new tool to further examine the tick-pathogen relationship. Taken together, this work uses a biologically relevant model to examine how the extrinsic incubation period allows for the perpetuation of tick-borne disease in nature.