Digital PCR (dPCR) shows great promise for precise, sensitive, and inhibition-resilient measurement of nucleic acids in surface water, providing an advantage for applications such as microbial source tracking (MST). Herein, we describe our empirical optimization of two triplex format dPCR reactions for MST (Triplex 1—Cow M2, Rum2Bac, Cow M3 and Triplex 2—HF183/BacR287, Pig2Bac, Entero1a) on the QIAcuityDeviceQIAcuity One system. Each triplex provides gene copy measurements similar to those obtained from single-plex format assays for a standard reference material (SRM 2917) at a fixed concentration and along a concentration gradient. On water samples previously tested by single-plex assays, each triplex also produces MST marker measurements comparable to the single-plex results. The triplexes we describe here can be directly adopted for MST on the QIAcuityDeviceQIAcuity, or the optimization protocol we demonstrate can be used to develop additional multiplex assays on the QIAcuityDeviceQIAcuity system.