A collection of 70 Burkholderia cepacia complex isolates, recovered from clinical, water, and agricultural resources in China in our previous studies, were tested to assess their potential pathogenicity and association of biofilm formation with pathogenicity. The pathogenicity was tested in the alternative infection models alfalfa, detached lettuce midrib, Galleria mellonella (wax moth), rat agar bead, and lettuce intact leaves. Severe to moderate pathogenicity were observed for isolates of clinical and water origin compared to agricultural isolates, with the exception of a few clinical isolates exhibiting reduced pathogenicity. Virulent isolates persisted in rat lungs until 21 days post infection causing histopathological changes like inflammation, while in lettuce midrib tissues invasion, localization, and replication of bacteria were observed. Biofilm formation ability was also documented in high frequency among water and clinical virulent isolates compared to agricultural isolates. Although variations in pathogenicity were observed for a few isolates, results obtained from different model systems including lettuce were consistent. Our studies indicate that water and clinical isolates showed severe virulence and strong biofilm formation ability compared to agricultural isolates. The results also show lettuce as a promising infection model not only to study the pathogenicity factors used by Bcc bacteria but also for characterization the in vivo transcriptional profile for different niches adaptation of this opportunistic pathogen.