Diagnostic stewardship (DxS) for infectious disease testing requires a multi-disciplinary approach to optimize test selection, performance, interpretation and patient treatment. Nucleic acid amplification-based tests for the diagnosis of infectious diseases, or “molecular microbiology tests,” have rapidly expanded over the past two decades. With the increased availability and complexity of these tests, there is also an increased need for collaborative approaches to optimize test use to promote positive impacts on patient care, while mitigating potential negative impact or resource waste. In this review, we provide recommendations on building collaborative DxS teams, including microbiologists and the diverse stakeholders that use and interpret molecular microbiology tests. We then detail approaches to identify high-priority molecular microbiology tests that may need utilization assessment, select appropriate diagnostic stewardship interventions, and monitor the impact of implemented interventions. This strategic process may be employed by laboratories to realize optimal testing for selected tests at their institution.