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Objective: In 2002, New South Wales (NSW) Health introduced an updated policy for occupational screening and vaccination against infectious diseases. This study describes healthcare worker (HCW) immunity to hepatitis B, measles, mumps, rubella (MMR) and varicella based on serological screening, following introduction of this policy. Methods: HCW screening serology performed at two healthcare facilities in south western Sydney (Bankstown and Fairfield) was extracted for the period September 2003 to September 2005. Immunity to hepatitis B, MMR and varicella was quantitated and cross-tabulated against age, sex and staff risk category. Results: A total of 1,320 HCWs were screened. Almost two thirds were immune to hepatitis B while immunity to MMR and varicella ranged from 88% to 94%. Age stratification showed lower levels of measles immunity in those born after 1965. Conclusions: Despite availability of vaccination for over two decades, a significant proportion of HCWs at these two facilities were non-immune to hepatitis B. This is of concern for those non-immune staff involved in direct clinical care, who are at risk of blood and body fluid exposures. The small group of HCWs non-immune to MMR and varicella pose a risk to themselves and others in the event of an outbreak. Implications: There is a need for improved implementation of the occupational screening and vaccination policy, including better education of HCWs about the risks of non-immunity to vaccine preventable diseases. The revised 2007 NSW Health policy may assist this process and will need evaluation to determine whether HCW immunity improves in the coming years.