Successful interactions with the human respiratory epithelial mucosa are crucial for the pathogenesis and persistence of Haemophilus influenzae (Hi) infections. While the role of adhesins and surface-exposed proteins in host interactions has been extensively studied, the metabolic processes fueling pathogen survival in the host remain less understood. Ribose, a pentose sugar and nucleoside component, is a preferred Hi growth substrate. In vitro growth studies on ribose uptake (rbsB) and utilization (rbsK, rpiA) mutant strains revealed transport (rbsB) and phosphorylation (rbsK) are essential for ribose utilization. rbsB and rbsK mutant strains also showed reduced growth with nucleosides for which 1H-NMR metabolomics revealed the preferential use of ribose moiety as a carbon source. Only the rbsB mutant strain exhibited increased biofilm formation, a virulence-related property, by approximately 35%. As biofilm formation is controlled by Quorum Sensing (QS), and rbsB has a proposed role in sensing the autoinducer-2 (AI-2) signal, we tested AI-2 production. While AI-2 production was similar in the rbsB mutant and the WT strains under standard growth conditions, it decreased by ~50% under the oxidative stress in the mutant strain. Moreover, rbsB and rbsK mutants demonstrated reduced survival in mouse macrophage infection assays (3.1- and 7-fold reduction, respectively). No phenotypic changes were detected in 16HBE14 tissue cell infections. However, IL-8 production in 16HBE14 infections with the rbsB mutant strain was reduced by ~ 30%, possibly due to LOS modifications induced by impaired QS. Our results indicate while ribose is a preferred carbon source for Hi, ribose utilization is not critical in most infection settings (probably for the presence of glucose in the medium), except where high levels of oxidative stress are present. Intriguing is the link of ribose transport to QS and oxidative stress resistance, that will need to be explored further and could be linked to altered LOS composition.