Burkholderia cenocepacia is a Gram-negative opportunistic pathogen belonging to the Burkholderia cepacia complex (Bcc). It is spread in a wide range of ecological niches, and in cystic fibrosis patients, it is responsible for serious infections. Its eradication is very difficult due to the high level of intrinsic resistance to clinically relevant antibiotics. One of the main resistance mechanisms in clinical isolates is represented by efflux systems that are able to extrude a variety of molecules, such as antibiotics, out of the cell. Resistance-Nodulation-Cell Division (RND) efflux pumps are known to be mediators of multidrug resistance in Gram-negative bacteria. Since now, the significance of the RND efflux systems in B. cenocepacia has been partially determined. However, the analysis of the completely sequenced genome of B. cenocepacia J2315 allowed the identification of 16 operons coding for these transporters. We focused our attention on the role of these pumps through the construction of several deletion mutants. Since manipulating B. cenocepacia J2315 genome is difficult, we used a peculiar inactivation system, which enables different deletions in the same strain. The characterization of our mutants through transcriptome and phenotype microarray analysis suggested that RND efflux pumps can be involved not only in drug resistance but also in pathways important for the pathogenesis of this microorganism. The aim of this review is an updated overview on host-pathogen interactions and drug resistance, particularly focused on RND-mediated efflux mechanisms, highlighting the importance of molecular techniques in the study of B. cenocepacia.