Although microbial risk due to antibiotic-resistant microbes in water has been a serious public health concern, the current culture-dependent detecting methods using nutrient-rich conditions may not be suitable for evaluating microbial risk. In the present study, a novel cultivation method was developed to detect antibiotic-resistant microbes at various nutrient levels. A nutrient-rich medium Luria-Bertani broth (LB) was diluted at a wide range of dilution factors (100−104) and amended with either tetracycline or vancomycin. A standard environmental cultivation medium, R2A, was also used by amending with antibiotics. The diluted LB with antibiotics was able to detect previously known oligotrophic and antibiotic-resistant bacteria in drinking water and swine manure samples, respectively. These results validate the capability of the new method to detect antibiotic-resistant microbes in various environmental water samples. Using the developed method in assessing microbial risk due to antibiotic-resistant microbes in river and municipal wastewater plants, we found that the viable counts and antibiotic resistance fraction were significantly influenced by the type and concentration level of antibiotic exposure and the cultivation medium conditions. This suggests a further need to standardize cultivation method procedures, to assess microbial risk in water samples.