The opportunistic nosocomial pathogen Acinetobacter baumannii is responsible for a growing number of infections; however, few of its potential virulence factors have been identified, and how this organism causes infection remains largely unknown. Bacterial biofilms are often an important component in infection and persistence but there is no conclusive evidence to link biofilm formation with virulence and severity of infection in Acinetobacter. To investigate this link, several clinical isolates were assessed in biofilm culture models and were tested for virulence in the insect model Galleria mellonella. In both systems, the profiles showed significant differences between strains, but no correlation was observed between virulence and the ability to form biofilms. In contrast, A. baumannii cells from a biofilm produced higher mortality rates than an equivalent number of planktonic cells. Relative to planktonic cells, A. baumannii biofilm cultures also showed reduced sensitivity to antibiotics normally used in the treatment of A. baumannii, especially colistin. This model, therefore, provides a suitable system to investigate the link between biofilm growth and various factors influencing virulence during A. baumannii infection.