Quantitative polymerase chain reaction (qPCR) is now commonly used to detect fecal indicator bacteria (FIB) as well as pathogens in water samples. However, DNA loss during sample processing can cause underestimation of target genes. In this study, we created a sample process control strain (SPC) by genetically engineering a non-pathogenic, Gram-negative bacterium Pseudogulbenkiania sp. strain NH8B. The SPC strain, named NH8B-1D2, has a kanamycin-resistance gene inserted to one of the 23S rRNA genes. To specifically quantify the SPC strain, a new TaqMan qPCR assay was developed. To obtain the relationship between the DNA recovery efficiencies of various pathogens and those of the SPC strain, known amount of E. coli O157:H7, Salmonella Typhimurium, Campylobacter jejuni, or Listeria monocytogenes cells were co-spiked with the SPC strain to environmental water samples. The DNA recovery efficiencies were calculated by comparing the quantity of bacterial cells inoculated to water samples prior to filtration and DNA extraction, and those measured by qPCR. We then obtained the ratios in the recovery efficiencies between pathogens and SPC strain (RRPATH/SPC). The RRPATH/SPC values obtained using Oono pond water collected in Japan were used as a pathogen-specific constant to estimate the accurate concentrations of pathogens in water samples collected from Mississippi River in Minnesota. Estimated pathogen concentrations were not significantly different from the inoculated pathogen concentration, suggesting our normalization approach is useful to estimate the accurate concentrations of pathogens in environmental water samples. The qPCR assay targeting the SPC strains and FIB were incorporated into the microfluidic qPCR chip format (PBQ chip ver. 2); therefore, we can simultaneously quantify multiple pathogens, FIB, and the SPC strain in high throughput from many water samples. This new tool can be useful for water quality monitoring and risk assessment.