We investigated the persistence of feces-derived Bacteroidales cells and their DNA in seawater under natural conditions using an optimized chemical method based on co-extraction of nucleic acids with propidium monoazide (PMA), which interferes with PCR amplification of molecular markers from extracellular DNA and dead bacterial cells. The previously validated Bacteroidales assays BacUni-UCD, BacHum-UCD, BacCow-UCD, and BacCan-UCD were utilized to determine concentrations of Bacteroidales genetic markers targeting all warm-blooded animals, humans, cows and dogs, specifically, over a period of 24 d. Microcosms containing mixed feces in dialysis tubing were exposed to seawater under flow-through conditions at ambient temperature in the presence and absence of sunlight. Using a two-stage plus linear decay model, the average T 99 (two-log reduction) of host-specific Bacteroidales cells was 28 h, whereas that of host-specific Bacteroidales DNA was 177 h. Natural sunlight did not affect the survival of uncultivable Bacteroidales cells and their DNA with the exception of the BacCow-UCD marker. Bacteroidales DNA, as measured by quantitative PCR (qPCR) without PMA, persisted for as long as 24 d at concentrations close to the limit of detection. Culturable Enterococcus cells were detected for only 70 h, whereas Enterococcus cells measured by qPCR with and without PMA persisted for 450 h. In conclusion, measuring Bacteroidales DNA without differentiating between intact and dead cells or extracellular DNA may misinform about the extent of recent fecal pollution events, particularly in the case of multiple sources of contamination with variable temporal and spatial scales due to the relatively long persistence of DNA in the environment. In contrast, applying qPCR with and without PMA can provide data on the fate and transport of fecal Bacteroidales in water, and help implement management practices to protect recreational water quality.