Introduction: Healthcare workers (HCWs), especially cancer care teams, are at an increased risk of SARS-CoV-2 infection, which may be amplified in rural areas. Therefore, we aimed to determine SARS-CoV-2 seroprevalence among HCWs in our rural cancer care network. Methods: We conducted a multisite, longitudinal, three-part SARS-CoV-2 seroprevalence study within the MaineHealth Cancer Care Network between November 2020 and April 2023, spanning metropolitan (one site), micropolitan (four), and rural (six) practices in south-central Maine and eastern New Hampshire. Follow-up was completed in January 2024. We followed 435 HCWs who provided cancer care during the pandemic. Participants reported their own medical history and that of household members and job duties, completed Occupational Risk and End-of-Study surveys, and underwent serial SARS-CoV-2 antibody testing. Results: In Part 1, the primary outcome SARS-CoV-2 period seroprevalence rate was 1.6% (3/192; 95% CI 0.5–4.5%). Nearly 8% of participants and 10% of their household members reported immunocompromising conditions. We observed persistence of spike protein antibody response >2,500 U/mL over the course of the study. Survey response rates were high and remarkably consistent, given the diversity of participants. Outside the workplace, our care team members practiced a high level of personal safety measures during the pandemic. Conclusion: Our findings showed a low SARS-CoV-2 seroprevalence, consistent with the national and international data reported early in the pandemic. We also observed higher-than-expected immunocompromising conditions in rural HCWs and their households. These results underscore the importance of longitudinal sero-surveillance, particularly for high-risk rural healthcare networks.