As the National Plant Protection Organisation laboratory, the Plant Health and Environment Laboratory (PHEL) is responsible for providing advice on pest status, reviewing the list of regulated plant pests, and verifying the identity of organisms suspected to be new for Aotearoa New Zealand. Fungal and bacterial reference collections and their associated metadata are important in this process, as they help to determine which plant pathogens are already present in the country, and the host plants they are associated with. This information is particularly important during a suspected incursion, when it is critical to determine if the organism of interest has previously been found in the area. We present three examples that illustrate the value of reference collections for decision-making in biosecurity: i) a rust fungus suspected to be a new detection, which was proven to have been present for many years through analysing DNA sequences of herbarium specimens; ii) a bacterium that was thought absent from Aotearoa New Zealand, but was proven to be present when analysing historical bacterial isolates collected across different geographic regions and several years; and iii) a Fusarium species – suspected as a first detection – which was shown not to be novel by analysing DNA sequences of cultures that were collected before the species was described. These examples highlight the value of comprehensive and accessible collections for assessing the risks which a new detection might pose, and for making decisions on appropriate biosecurity measures. PHEL uses both in-house and publicly available collections to support their diagnostic activities, which are instrumental to support Aotearoa New Zealand’s biosecurity and international trade.