Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen associated with a range of infections, including various lower respiratory infections, otitis media, and conjunctivitis. There is some debate as to whether or not NTHi produces biofilms and, if so, whether or not this is relevant to pathogenesis. Although many studies have examined the association between in vitro biofilm formation and isolates from a specific infection type, few have made comparisons from isolates from a broad range of isolates grouped by clinical source. In our study 50 NTHi from different clinical sources, otitis media, conjunctivitis, lower respiratory tract infections in both cystic fibrosis and non-cystic fibrosis patients, and nasopharyngeal carriage, plus 10 nasopharyngeal isolates of the commensal Haemophilus haemolyticus were tested for the ability to form biofilm by using a static microtitre plate crystal violet assay. A high degree of variation in biofilm forming ability was observed across all isolates, with no statistically significant differences observed between the groups, with the exception of the isolates from conjunctivitis. These isolates had uniformly lower biofilm forming ability compared with isolates from the other groups (p < 0.005).