Tuberculosis (TB) kills more individuals globally than any other single infectious disease. Health care professionals and household contacts in endemic areas have routinely high Mycobacterium tuberculosis (M.tb) pathogen exposures. Focused intervention strategies designed to block transmission in these populations would help to significantly reduce the burden of TB disease. Mycobacteriophages (phage) are an underutilized biologic intervention for the pathogen M.tb and could be leveraged for their bactericidal properties. Our goal was to address outstanding questions about the utility of phage therapy against M.tb. Specifically, we determined the impact of repeated mucosal or intravenous (i.v.) exposures to anti-M.tb phage FionnbharthΔ45Δ47 (Fionnbharth), an engineered lytic phage on host anti-phage immunity in a preclinical mouse model. We hypothesized that repeated i.v. phage delivery would induce high titers of functionally neutralizing anti-phage antibodies, but mucosal delivery via the aerosol route would not. To evaluate the impact of repeat exposures on host anti-phage immunity, male and female C57BL/6 mice were dosed 6 times weekly with Fionnbharth i.v. or via aerosol, and serum and bronchoalveolar lavage fluid (BALf) were collected weekly. The anti-phage antibody magnitude and endpoint titer (EPT) for each condition and timepoint was determined using an in-house developed ELISA. We observed that repeated i.v. phage dosing induced a significantly higher magnitude of anti-phage humoral responses than the same phage delivered via aerosol over the entire time period evaluated. After 6 weekly i.v. doses, anti-phage total IgG EPTs for serum samples reached a magnitude of 5.0 log10 while aerosol cohorts were often below or at the assay’s limit of detection (1.0 log10). Endpoint titers of anti-phage total IgG in BALf samples for i.v. dosed mice reached 2.0 log10, while the aerosol cohorts remained below the limit of detection (0.0 log10 EPT). We observed a reverse profile for IgA samples where aerosol treated cohorts had a low but detectable serum EPT (2.1 log10) and i.v. cohorts were at the limit of detection (1.0 log10). Importantly, we demonstrated that serum samples collected after 6 weeks of repeated i.v. dosing contained functionally neutralizing antibodies that were able to inhibit phage-mediated lysis of a representative mycobacteria, M. smegmatis. We did not observe the same phenotype from aerosol treated cohort samples. These data suggest that aerosol delivery of phage is a viable approach for therapy because it does not induce a robust neutralizing anti-phage response and should be considered as a prioritized route for further evaluation of efficacy against M.tb.