Human blood components have been shown to enhance biofilm formation by gram-positive bacteria. We investigated the effect of human blood on biofilm formation on the inner lumen of needleless central venous catheter connectors by several gram-negative bacteria, specifically Enterobacter cloacae, Pseudomonas aeruginosa, and Pantoea agglomerans. Results suggest that a conditioning film of blood components promotes biofilm formation by these organisms in an in vitro system.