There is little information on the indoor environment in hotels. Analysis of fungal DNA by quantitative PCR (qPCR) is a new method which can detect general and specific sequences. Dust was collected through swab sampling of door frames in 69 hotel rooms in 20 countries in Europe and Asia (2007–2009). Five sequences were detected by qPCR: total fungal DNA, Aspergillus and Penicillium DNA (Asp/PenDNA), Aspergillus versicolor (A. versicolorDNA), Stachybotrys chartarum (S. chartarumDNA) and Streptomyces spp. (StreptomycesDNA). Associations were analysed by multiple linear regression. Total fungal DNA (GM = 1.08 × 10⁸cell equivalents m⁻²; GSD = 6.36) and Asp/PenDNA (GM = 1.79 × 10⁷cell equivalents m⁻²; GSD = 10.12) were detected in all rooms. A. versicolorDNA, S. chartarumDNA and StreptomycesDNA were detected in 84%, 28% and 47% of the samples. In total, 20% of the rooms had observed dampness/mould, and 30% had odour. Low latitude (range 1.5–64.2 degrees) was a predictor of Asp/PenDNA. Seaside location, lack of mechanical ventilation, and dampness or mould were other predictors of total fungal DNA and Asp/PenDNA. Hotel ranking (Trip Advisor) or self-rated quality of the interior of the hotel room was a predictor of total fungal DNA, A. versicolorDNA and StreptomycesDNA. Odour was a predictor of S. chartarumDNA. In conclusion, fungal DNA in swab samples from hotel rooms was related to latitude, seaside location, ventilation, visible dampness and indoor mould growth. Hotels in tropical areas may have 10–100 times higher levels of common moulds such as Aspergillus and Penicillium species, as compared to a temperate climate zone.