Chryseobacterium indologenes and Chryseobacterium gleum are Gram negative environmental bacteria that have been frequently reported to implicate in fatal nosocomial infections, such as bacteraemia and ventilator-associated pneumonia in immunocompromised individuals in the past decades. The interaction between Chryseobacterium spp. and Acanthamoeba castellanii, a free-living amoeba ubiquitous in the environment, has not been explored previously. In this study, C. indologenes and C. gleum were co-cultured with A. castellanii trophozoites and their interactions were evaluated. Our results showed that when co-cultured with A. castellanii, bacterial numbers of C. indologenes and C. gleum increased significantly (p 0.05). Interestingly, the two Chryseobacterium spp. associated, invaded and/or taken up by A. castellanii at significantly higher rates than Escherichia coli K1, a neuropathogenic bacterial strain known to interact and replicate intracellularly in A. castellanii (p