Cladosporium is one of the most common airborne molds found in indoor and outdoor environments. Cladosporium spores are important aeroallergens, and prolonged exposure to elevated spore concentrations can provoke chronic allergy and asthma. To accurately quantify the levels of Cladosporium in indoor and outdoor environments, two real-time PCR systems were developed in this study. The two real-time PCR systems are highly specific and sensitive for Cladosporium detection even in a high background of other fungal DNAs. These methods were employed to quantify Cladosporium in aerosols of five different indoor environments. The investigation revealed a high spore concentration of Cladosporium (10⁷ m⁻³) in a cow barn that accounted for 28–44% of the airborne fungal propagules. In a countryside house that uses firewood for heating and in a paper and pulp factory, Cladosporium was detected at 10⁴ spores m⁻³, which accounted for 2–6% of the fungal propagules in the aerosols. The concentrations of Cladosporium in these three indoor environments far exceeded the medical borderline level (3000 spores m⁻³). In a power station and a fruit and vegetable storage, Cladosporium was found to be a minor component in the aerosols, accounted for 0.01–0.1% of the total fungal propagules. These results showed that monitoring Cladosporium in indoor environments is more important than in outdoor environments from the public health point of view. Cladosporium may not be the dominant fungi in some indoor environments, but its concentration could still be exceeding the threshold value for clinical significance. The methods developed in this study could facilitate accurate detection and quantification of Cladosporium for public health related risk assessment.