The activity of biocides against Aspergillus fumigatus is unknown. In theEuropean guidelines to evaluate the fungicidal activity of a biocide, thecritical step concerning the preparation of conidial suspensions iscumbersome and time-consuming. The aims of this study were to evaluate asimplified procedure to prepare conidial suspensions to test a biocide incomparison with the recommended one and to investigate the in vitro activityof seven biocides by the suspension neutralization method against A.fumigatus clinical isolates. The proposed simplified procedure provedreproducible, gave the same results and was quicker than that described inthe European guidelines. Benzalkonium chloride (0.25 %), glutaraldehyde (1.6%), polyvinylpyrrolidone iodine (1 % available iodine) and polyester glycoliodine (0.18 % available iodine) showed biocidal activity in <or=5 mincontact time, and chlorine (0.14 % available Cl) and chlorhexidine (0.06 %)after 15 min and 30-60 min, respectively. In contrast, chloramine-T (0.01 %)did not show biocidal activity. In addition, a simplified and reproducibleprocedure may be used for testing the fungicidal activity of new compoundsor combined formulations. In conclusion, the biocides tested, which arecommonly used in hospital settings, were shown to display biocidal activityagainst A. fumigatus and a simplified procedure may be adopted for testingthe fungicidal activity of new compounds.