Quantitative polymerase chain reaction (qPCR) offers a rapid, highly sensitive analytical alternative to the traditional culture-based techniques of microbial enumeration typically used in water quality monitoring. Before qPCR can be widely applied within surface water monitoring programs and stormwater assessment research, the relationships between microbial concentrations measured by qPCR and culture-based methods must be assessed across a range of water types. Previous studies investigating fecal indicator bacteria quantification using molecular and culture-based techniques have compared measures of total concentration, but have not examined particle-associated microorganisms, which may be more important from a transport perspective, particularly during the calibration of predictive water quality models for watershed management purposes. This study compared total, free-phase, and particle-associated Escherichia coli concentrations as determined by the Colilert defined substrate method and qPCR targeting the uidA gene in stream grab samples partitioned via a calibrated centrifugation technique. Free-phase concentrations detected through qPCR were significantly higher than those detected using Colilert although total concentrations were statistically equivalent, suggesting a source of analytical bias. Although a specimen processing complex was used to identify and correct for inhibition of the qPCR reaction, high particle concentrations may have resulted in underestimation of total cell counts, particularly at low concentrations. Regardless, qPCR-based techniques will likely have an important future role in stormwater assessment and management.