In order to understand microbial diversity-function relationship, direct quantification of their phenotypes is important as well as omics approaches. Here, we aimed to demonstrate the utility of BIOLOG microplate to evaluate the capability of microbial communities to decompose diverse carbon substrates in two systems: a freshwater system (Feitsui reservoir in Taipei) and a bamboo forest soil system (Xitou experimental forest in Nantou). From the color development patterns of each microplate, we calculated the indices for functional dissimilarity, functional diversity (multifunctionality), and bulk functional potential. From the freshwater system, we found that the functional predictability (evaluated by variations between replicates) increased with functional diversity of bacteria but decreased with temperature. From the soil system, functional predictability of bacteria (or fungi) decreased (or increased) with bacterial (or fungal) functional diversity. In addition, the functional dissimilarity of bacteria was positively correlated with that of fungi. These results indicated the effectiveness of the BIOLOG method for studying microbial diversity and ecosystem functions.