Summary Heating, ventilation and air conditioning (HVAC) systems make it possible to maintain good indoor air quality in different types of buildings. Without proper maintenance, however, these systems can become sources of emission of air pollutants. Today, decisions about when air duct cleaning is required are often based on dust fall levels. However, the fungal biomass of the dust should also be considered, because the amount of dust deposited is not in any way indicative of its microbial load. To take into account all possible health effects, two criteria must be considered for proper evaluation of air duct cleanliness: dust fungal biomass and dust weight. The purpose of this project is to help building managers make informed decisions about the cleanliness of HVAC systems and thus prevent the exposure of building occupants to mould. Project objectives were twofold: 1) to compare two methods of collecting dust deposited in HVAC ductwork, evaluating their performance and limitations; and 2) to fine-tune, validate and compare methods of determining the fungal biomass of the dust. The first part of the project consisted in determining the best method of collecting dust deposited in HVAC systems. One of the two methods assessed, involving wiping, was rapidly abandoned, because it was difficult to collect dust this way from the smooth surfaces of the ductwork. The dust collection efficiency of the other method, involving suction with an Environmental Monitoring Systems (EMS) cassette with integrated nozzle, proved to be excellent—more than 98% when the entire cassette was processed. In the second part of the project, fungal biomass analysis methods demonstrating acceptable performances when validated were compared in a controlled environment as well as in the field. The universal qPCR assay proved simple, rapid and effective, with its very low detection limit and a processing time of less than 24 hours. Given its performance, low-cost and short analysis time, the universal qPCR is currently the most suitable method for evaluating air duct cleanliness. In addition to providing an analytical method for assessing fungal biomass, this project also demonstrates that dust fungal load as well as dust weight must be considered in assessing air duct cleanliness.