This study developed, optimized, and demonstrated a microfluidic quantitative polymerase chain reaction (MF-qPCR) method for the simultaneous quantification of 39 antibiotic resistance genes (ARGs), five heavy metal resistance genes, three genes encoding the integrase of three different classes of integrons, and 16S rRNA genes (used as a measure of total bacterial biomass). Because the volume of the template is much smaller with MF-qPCR (a few nanoliters) than with conventional qPCR, a preamplification step was needed to improve the sensitivity and the limits of quantification of the MF-qPCR method to be similar to those of conventional qPCR. The MF-qPCR method was successfully demonstrated on untreated municipal wastewater, treated municipal wastewater, and drinking water samples. The treated municipal wastewater samples had higher concentrations of all genes compared to those in the drinking water samples. Similarly, the untreated municipal wastewater samples had higher concentrations for all but one of the targeted genes compared to those in the treated municipal wastewater samples. The MF-qPCR method established in this study provides highly accurate quantitative information about numerous ARGs and other genes from environmental samples.