The pressing need for novel chemical matter to support bioactive compound discovery has led natural product researchers to explore a wide range of source organisms and environments. One of the implicit guiding principles behind those efforts is the notion that sampling different environments is critical to accessing unique natural products. This idea was tested by comparing fungi from disparate biomes: aquatic sediments from Lake Michigan (USA) and terrestrial samples taken from the surrounding soils. Matched sets of Penicillium brevicompactum, Penicillium expansum, and Penicillium oxalicum from the two source environments were compared, revealing modest differences in physiological performance and chemical output. Analysis of LC-MS/MS-derived molecular feature data showed no source-dependent differences in chemical richness. High levels of scaffold homogeneity were also observed with 78-83% of scaffolds shared among the terrestrial and aquatic Penicillium spp. isolates. A comparison of the culturable fungi from the two biomes indicated that certain genera were more strongly associated with aquatic sediments (e.g., Trichoderma, Pseudeurotium, Cladosporium, and Preussia) versus the surrounding terrestrial environment (e.g., Fusarium, Pseudogymnoascus, Humicola, and Acremonium). Taken together, these results suggest that focusing efforts on sampling the microbial resources that are unique to an environment may have a more pronounced effect on enhancing the sought-after natural product diversity needed for chemical discovery and screening collections.