Though nitrogen dynamics have been described for stream ecosystems, few studies have characterized microbial communities responsible for nitrogen cycling in streams. This study described microbial communities in Fossil Creek based on functional genes involved in nitrogen cycling: nifH for nitrogen fixation and bacterial and archaeal amoA for nitrification. Communities were compared among water and substrate samples in travertine terrace and riffle-pool reaches using relative gene abundance from real-time polymerase chain reaction. For all water and substrate samples, archaeal amoA genes were present, though bacterial amoA was not detected. However, 16S ribosome genes for ammonia oxidizing bacteria (AOB) were present in all samples indicating that an alternate amoA primer set is likely needed to target freshwater AOB functional genes. Results suggest that archaeal ammonia oxidizers play important roles in nitrification in streams. We found greater abundances of archaeal amoA, AOB 16S, and nifH in the travertine terrace reach compared with the riffle-pool reach. This suggests that nitrification and nitrogen fixation rates are higher in travertine reaches. There were no significant differences in abundances of these genes between substrates within each reach which likely reflect similar community structures despite unique microhabitats.