The failing eradication of bacterial biofilm by antibiotic treatment is a major clinical challenge for patients with chronic infections. The effect of re-sensitizing infectious biofilms to antibiotics by supplemental O2 has recently been explained by the pathophysiological conditions of the infectious microenvironment. In this chapter, we argue that multifactorial restrictions of O2, caused by O2 depletion within the infectious microenvironment, determine the tolerance of bacterial phenotypes ranging from the endogenous formation of persistent subpopulations within biofilms to the induction of tolerant biofilm by the host response. Consequently, we describe how reoxygenation may re-sensitize microorganisms growing in biofilms to antibiotics. To further emphasize the importance of O2 distribution we demonstrate the significance of O2 availability for the outcome of susceptibility testing.