Outbreaks of pseudo-infection due to contamination of specimens have been described, often as localised incidents. From August 2006, several English hospital laboratories began to refer an unusually high number of isolates of the fungus Paecilomyces variotii from clinical specimens to the national mycology reference laboratory for microbiological testing. We describe the methods used during the outbreak investigation in order to provide infection control specialists with an overview of how such national incidents may be investigated. We surveyed the hospitals reporting the contamination problem and conducted microbiological and environmental sampling. We applied analytical epidemiology to supply chain data, comparing the supply lines of key equipment to affected and unaffected hospitals in England. The survey was useful to describe procedures and equipment in use in the hospitals reporting the problem. The microbiological aspects of the investigation helped us understand how the fungal spores were distributed in the hospital environment. In the supply chain investigation we used data that was previously only used for logistical purposes. Overall the investigation was methodologically challenging, with no existing protocol to guide the investigators. To our knowledge, this is a novel approach to the investigation of such a widespread contamination problem, affecting geographically disparate hospitals at the same time.