This study aimed at evaluating the host-specificity and -sensitivity of the nifH gene marker of Methanobrevibacter smithii by screening 272 fecal and wastewater samples from 11 animal species including humans in Southeast Queensland (SEQ), Australia. In addition, environmental water samples (n = 21) were collected during the dry and wet weather conditions and tested for the presence of the nifH marker along with other sewage-associated markers, namely, enterococci surface protein (esp) found in Enterococci faecium, Bacteroides HF183, adenoviruses (AVs), and polyomaviruses (PVs). The overall host-specificity of the nifH marker to differentiate between human and animal feces was 0.96 (maximum value of 1), while the overall sensitivity of this marker in human sourced feces and wastewater was 0.81 (maximum value of 1). Among the 21 environmental water samples tested, 2 (10%), 3 (14%), 12 (57%), 6 (29%), and 6 (29%) were positive for the nifH, esp, HF183, AVs and PVs markers, respectively. The prevalence of the nifH marker in environmental water samples, however, was low compared to other markers, suggesting that the use of this marker alone may not be sensitive enough to detect fecal pollution in environmental waters. The nifH marker, however, appears to be sewage-specific in SEQ, Australia, and therefore, it is recommended that this marker should be used as an additional marker in combination with the HF183 or viral markers such as AVs or PVs for accurate and sensitive detection of fecal pollution in SEQ waterways.