Graywater (GW) reuse for irrigation is recognized as a sustainable solution for water conservation. One of the major impediments to GW reuse is the presence of pathogenic microorganisms. This study monitored three similar on-site GW treatment systems bi-monthly over the course of a year to compare the presence of pathogens and indicators in raw, biologically treated, and biologically treated and disinfected [by chlorine and ultraviolet light (UV)] GW. The systems were designed to allow the testing of the same batch (collection) of water as it passed through the treatment chain. The samples were analyzed using standard culture-dependent methods and the data were compared to culture-independent DNA-based methods. Results suggested that the presence and abundance of fecal coliforms, Escherichia coli, Salmonella enterica, Enterococcus spp., Staphylococcus aureus and Pseudomonas aeruginosa differ among the various GW streams (e.g. raw, biologically treated, and disinfected). The culture-dependent analyses suggested that both chlorine and UV inactivate most of the bacteria tested in the biologically treated GW, albeit at different efficiencies. Conversely, the DNA-based analyses indicated no significant differences in pathogenic bacterial abundance between the biologically treated GW with or without disinfection. To better understand the discrepancies between the results, we repeated the analysis in the laboratory under controlled conditions using Enterococcus faecalis as a model bacterium and obtained similar results. We suggest that disinfection of biologically treated GW with chlorine or UV is effective for treating pathogens, but that the inactivation efficiency cannot be estimated by DNA-based qPCR.