Pathogenic microorganisms are responsible for many infectious diseases, and pathogen monitoring is important and necessary for water quality control. This study for the first time explored a multiplex quantitative real-time PCR (qPCR) technique combined with propidium monoazide (PMA) to simultaneously detect viable Legionella pneumophila, Salmonella typhimurium, and Staphylococcus aureus in one reaction from water samples. Sodium lauroyl sarcosinate (sarkosyl) was applied to enhance the dead bacterial permeability of PMA. The sensitivity of the multiplex PMA-qPCR assay achieved two colony-forming units (CFU) per reaction for L. pneumophila and three CFU per reaction for S. typhimurium and S. aureus. No PCR products were amplified from all nontarget control samples. Significantly, with comparable specificity and sensitivity, this newly invented multiplex PMA-qPCR assay took a much shorter time than did conventional culture assays when testing water samples with spiked bacteria and simulated environmental water treatment. The viable multiplex PMA-qPCR assay was further successfully applied to pathogen detection from rivers, canals, and tap water samples after simple water pretreatment.