We aimed to detect physiological characteristics that clearly varied among the closely-related Cladosporium sphaerospermum-like species. We isolated the fungi identified as C. sphaerospermum s.l. based on traditional morphological criteria from various locations and substrata, and redefined this initial identification by the molecular phylogenetic methods. The isolates were identified as only C. sphaerospermum and C. halotolerans. We analyzed the substrate-utilization of 95 carbon sources using the Biolog system and made statistical comparisons of isolates by their abilities to grow at different osmolarities. The substrate-utilization patterns separated the isolates into two groups corresponding to the molecular data, and the osmotolerance was different between the species. We first showed that C. sphaerospermum and C. halotolerans were diverse not only at the molecular level but also at the ecological and the physiological levels, by analyzing substrate-utilization patterns and osmotolerance. Furthermore, we showed the potential utility of the Biolog system for discriminating among closely-related fugal species.