Burkholderia cenocepacia is an important human pathogen in patients with cystic fibrosis (CF). Non-clinical reservoirs may play a role in the acquisition of infections, so it is important to evaluate the pathogenic potential of environmental B. cenocepacia isolates. In this study, we investigated the interactions of two environmental B. cenocepacia strains (Mex1 and MCII-168) with two bronchial epithelial cell lines, 16HBE14o- and CFBE41o-, which have a non-CF and a CF phenotype, respectively. The environmental strains showed a significantly lower level of invasion into both CF- and non-CF cells in comparison with the clinical B. cenocepacia LMG16656(T) strain. Exposure of polarized CFBE41o- or 16HBE14o- cells to the environmental strains resulted in a significant reduction in transepithelial resistance (TER), comparable to that observed following exposure to the clinical strain. A different mechanism of tight junction disruption in CF versus non-CF epithelia was found. In the 16HBE41o- cells, the environmental strains resulted in a drop in TER without any apparent effect on tight junction proteins such as zonula occludens-1 (ZO-1). In contrast, in CF cells, the amount of ZO-1 and its localisation were clearly altered by the presence of both the environmental strains, comparable to the effect of LMG16656. This study demonstrates that even if the environmental strains are significantly less invasive than the clinical strain, they have an effect on epithelial integrity comparable to that of the clinical strain. Finally, the tight junction regulatory protein ZO-1 appears to be more susceptible to the presence of environmental strains in CF cells than in the cells which express functional CFTR.