West Virginia prioritized SARS-CoV-2 vaccine delivery to nursing home facilities because of increased risk of severe illness in elderly populations. However, the persistence and protective role of antibody levels remain unclear. To examine the persistence of humoral immunity after COVID-19 vaccination and the association of SARS-CoV-2 antibody levels and subsequent infection among nursing home residents and staff. In this cross-sectional study, blood samples were procured between September 13 and November 30, 2021, from vaccinated residents and staff at participating nursing home facilities in the state of West Virginia for measurement of SARS-CoV-2 antibody (anti-receptor binding domain [RBD] IgG). SARS-CoV-2 infection and vaccination history were documented during specimen collection and through query of the state SARS-CoV-2 surveillance system through January 16, 2022. SARS-CoV-2 vaccination (with BNT162b2, messenger RNA-1273, or Ad26.COV2.S). Anti-RBD IgG levels were assessed using multivariate analysis to examine associations between time since vaccination or infection, age, sex, booster doses, and vaccine type. Antibody levels from participants who became infected after specimen collection were compared with those without infection to correlate antibody levels with subsequent infection. Among 2139 SARS-CoV-2 vaccinated residents and staff from participating West Virginia nursing facilities (median [range] age, 67 [18-103] years; 1660 [78%] female; 2045 [96%] White), anti-RBD IgG antibody levels decreased with time after vaccination or infection (mean [SE] estimated coefficient, -0.025 [0.0015]; P < .001). Multivariate regression modeling of participants with (n = 608) and without (n = 1223) a known history of SARS-CoV-2 infection demonstrated significantly higher mean (SE) antibody indexes with a third (booster) vaccination (with infection: 11.250 [1.2260]; P < .001; without infection: 8.056 [0.5333]; P < .001). Antibody levels (calculated by dividing the sample signal by the mean calibrator signal) were significantly lower among participants who later experienced breakthrough infection during the Delta surge (median, 2.3; 95% CI, 1.8-2.9) compared with those without breakthrough infection (median, 5.8; 95% CI, 5.5-6.1) (P = .002); however, no difference in absorbance indexes was observed in participants with breakthrough infections occurring after specimen collection (median, 5.9; 95% CI, 3.7-11.1) compared with those without breakthrough infection during the Omicron surge (median, 5.8; 95% CI, 5.6-6.2) (P = .70). In this cross-sectional study, anti-RBD IgG levels decreased after vaccination or infection. Higher antibody responses were found in individuals who received a third (booster) vaccination. Although lower antibody levels were associated with breakthrough infection during the Delta surge, no significant association was found between antibody level and infection observed during the Omicron surge. The findings of this cross-sectional study suggest that among nursing home residents, COVID-19 vaccine boosters are important and updated vaccines effective against emerging SARS-CoV-2 variants are needed.