Bifidobacteria are one of the main groups of bacteria found in the intestine of humans and other animals. They have been proposed as indicators to identify the source of fecal pollution, since certain Bifidobacterium spp. are found only in humans. However, species identification remains difficult, and has limited their practical application. In this study, a simple assay based on the ratio of sorbitol-fermenting bifidobacteria to total colony forming units on Human Bifido Sorbitol Agar (HBSA) medium was developed. Ratios greater than 0.2 were indicative of human fecal pollution while animal-derived slaughterhouse effluent had a ratio less than 0.05. This is a low-cost, easily implemented approach which may have wide applicability when characterizing recent and high concentrated fecal pollution.