Background: Aliarcobacter faecis and Aliarcobacter lanthieri are recently identified as emerging human and animal pathogens. In this paper, we demonstrate the development and optimization of two direct DNA-based quantitative real-time PCR assays using species-specific oligonucleotide primer pairs derived from rpoB and gyrA genes for A. faecis and A. lanthieri, respectively. Initially, the specificity of primers and amplicon size of each target reference strain was verified and confirmed by melt curve analysis. Standard curves were developed with a minimum quantification limit of 100 cells mL − 1 or g − 1 obtained using known quantities of spiked A. faecis and A. lanthieri reference strains in autoclaved agricultural surface water and dairy cow manure samples. Results: Each species-specific qPCR assay was validated and applied to determine the rate of prevalence and quantify the total number of cells of each target species in natural surface waters of an agriculturally-dominant and non-agricultural reference watershed. In addition, the prevalence and densities were determined for human and various animal (e.g., dogs, cats, dairy cow, and poultry) fecal samples. Overall, the prevalence of A. faecis for surface water and feces was 21 and 28%, respectively. The maximum A. faecis concentration for water and feces was 2.3 × 10 7 cells 100 mL-1 and 1.2 × 10 7 cells g − 1 , respectively. A. lanthieri was detected at a lower frequency (2%) with a maximum concentration in surface water of 4.2 × 10 5 cells 100 mL − 1 ; fecal samples had a prevalence and maximum density of 10% and 2.0 × 10 6 cells g − 1 , respectively.