This study evaluated knowledge, attitudes and behaviour regarding infection control of dental hygienists in Italy. Among the 185 responders to the self-administered mailed questionnaire, 91.3% agreed with the correct responses to the three questions on knowledge about infection prevention and control procedures chosen as an indicator of ‘good’ knowledge. However, 21% were uncertain whether, or disagreed that, dental instruments should be rinsed in water after contact with glutaraldehyde and 17.5% agreed that, or were uncertain whether, 10min contact with glutaraldehyde provided sterilization. Only 36.5% knew all the five oral manifestations of acquired immunodeficiency syndrome (AIDS) (acute periodontal problems, candidiasis, hairy leukoplakia, herpes simplex virus, Kaposi's sarcoma) and this knowledge was significantly higher in dental hygienists with a lower number of years of practice. More than two-thirds used a steam sterilizer or glutaraldehyde for appropriate times and temperatures for disinfection/sterilization of instruments and used appropriate surface disinfection procedures. The correct application of disinfection or sterilization methods for instruments was more likely in the older respondents and in those who attended continuing education courses on infection control. A positive attitude was reported by the majority of dental hygienists who agreed that guidelines should be maintained and applied and was significantly more likely in younger respondents. Only 57.9% routinely follow all recommendations for infection control practices and their use was significantly higher in the older respondents. Educational programmes are needed for improving knowledge about oral manifestations of AIDS in order to support dentists to provide early diagnosis and about the correct use of procedures and universal precautions for preventing infections.