Wheeze is a common symptom in preschool children. The role of bacteria, regulatory T (T(reg)) cells and their association with airway inflammation in preschool wheeze is largely unknown. We evaluated inflammatory markers in exhaled breath condensate (EBC), bacterial colonization and circulating T(reg) cells in preschool children with and without recurrent wheeze. We recruited 252 children (aged two to four years) with (N = 202) and without (N = 50) recurrent wheeze. EBC was collected using an efficient closed glass condenser. Inflammatory markers in EBC (Interleukin(IL)-2, IL-4, IL-8, IL-10, IL-13) were assessed using multiplex immunoassay. Nasal and throat swabs were analysed for presence of Streptococcus pneumoniae, Haemophilus (para)influenzae and Staphylococcus aureus. Proportions of T(reg) cells (CD4(+)CD25(high)CD127(-)) were quantified by flow cytometry. Recurrent wheezing children had elevated EBC levels of IL-2, IL-4, IL-10 and IL-13 compared to non-wheezers (odds ratio (95% confidence interval): 1.67 (1.23-2.27): 1.58 (1.15-2.18): 1.47 (1.14-1.90): 1.55 (1.16-2.06), p <0.05, respectively). Bacteria were frequently present in children with and without wheeze, with no difference in prevalence (16-52% versus 16-50%, respectively). Moreover, the proportion of T(reg) cells did not differ between both groups. Wheezing children with bacterial colonization did not significantly differ in exhaled levels of inflammatory markers or proportion of T(reg) cells compared to wheezing children without colonization. The analysis of EBC might serve as a helpful non-invasive tool to early assess airway inflammation in wheezing children. The various elevated exhaled inflammatory markers indicate increased airway inflammation in wheezing preschool children. In the presence of wheeze, we found no evidence for bacterial induced airway inflammation.