Background: Total Worker Health® programs represent a holistic approach for advancing worker safety, health, and well-being that combines occupational safety and health practices with other well-being policies and programs that are not traditionally linked. Total Worker Health requires new practices of interdisciplinary collaboration and programmatic coordination, accompanied with employee involvement and shared decision-making in the design and delivery of health-focused interventions. Pre-implementation assessment regarding organizational readiness is important to plan proactively for organizational “fit” when the program is introduced. This study examined potential implementation facilitators and barriers among five public healthcare facilities that had already agreed to participate in a study to implement and evaluate a participatory Total Worker Health program.Methods: A mixed-methods baseline assessment comprised an online organizational readiness survey and follow-up interviews. Key organizational and labor leaders were asked to identify resources and skills available for program implementation, potential barriers inside or outside the organization, and specific performance indicators. Findings at each facility were presented to implementation stakeholders and used to plan how to tailor implementation to the organizational context and provide resources necessary for success. Results: Implementation facilitators included leaders’ willingness and commitment to develop interventions addressing a broad range of occupational safety and well-being health priorities; existing staff expertise in occupational safety and health; favorable attitudes regarding expected program outcomes; and positive alignment between the program and organizational mission and values. Implementation barriers included limited staff time to attend meetings, limited resources to support locally designed interventions, and poor management communication systems. Examples of proactive tailoring strategies included extending the recruitment time and effort to gain the trust of leaders and workers while also allowing more time to secure needed program resources; developing sample program communication templates to strengthen communication on safety and health; and detailed training around health and safety issue identification and prioritization procedures, program communication, intervention planning and development, and change management. Conclusions: Prospective identification of potential program facilitators and barriers represents a useful strategy for tailoring implementation of a participatory Total Worker Health program. Trial Registration: ClinicalTrials.gov NCT 04251429. Registered January 31, 2020 – Retrospectively registered. https://clinicaltrials.gov/ct2/show/record/NCT04251429.