Background: Pseudomonas aeruginosa is difficult to eradicate from the lungs of cystic fibrosis (CF) patients due to biofilm formation. Organs and blood are independent pharmacokinetic (PK) compartments. Previously, we showed in vitro biofilms behave as independent compartments impacting the pharmacodynamics. The present study investigated this phenomenon in vivo. Methods: Seaweed alginate beads with P. aeruginosa resembling biofilms, either freshly produced (D0) or incubated for 5 days (D5) were installed s.c in BALB/c mice. Mice (n = 64) received tobramycin 40 mg/kg s.c. and were sacrificed at 0.5, 3, 6, 8, 16 or 24 h after treatment. Untreated controls (n = 14) were sacrificed, correspondingly. Tobramycin concentrations were determined in serum, muscle tissue, lung tissue and beads. Quantitative bacteriology was determined. Results: The tobramycin peak concentrations in serum was 58.3 (±9.2) mg/L, in lungs 7.1 mg/L (±2.3), muscle tissue 2.8 mg/L (±0.5) all after 0.5 h and in D0 beads 19.8 mg/L (±3.5) and in D5 beads 24.8 mg/L (±4.1) (both 3 h). A 1-log killing of P. aeruginosa in beads was obtained at 8h, after which the bacterial level remained stable at 16 h and even increased in D0 beads at 24 h. Using the established diffusion retardation model the free tobramycin concentration inside the beads showed a delayed buildup of 3 h but remained lower than the MIC throughout the 24 h. Conclusions: The present in vivo study based on tobramycin exposure supports that biofilms behave as independent pharmacological microcompartments. The study indicates, reducing the biofilm matrix would increase free tobramycin concentrations and improve therapeutic effects.