Background: During the COVID-19 pandemic, serological tests to screen populations have provided better estimates of the cumulative incidence of infection. This study evaluated the seroprevalence of SARS-CoV-2 in agricultural workers in rural Guatemala, their COVID-19 vaccine uptake and attitudes and hesitancy to vaccination. Methods: A cross-sectional study was undertaken of from August to November of 2021, in prospective agricultural workers at a sugar plantation in Guatemala. A standardized questionnaire was used to collect demographic, previous COVID-19 infection, vaccination, and attitudes toward vaccination. Serological testing was performed to detect SARS-CoV-2 IgM and IgG. Results: Of 4,343 workers consented and with a viable sample, 1,279 (29.4%) were seropositive for SARS-CoV-2 IgG and/or IgM compared to 2.3% reporting having COVID-19 infection in the past; 85% had received the first dose of the COVID-19 vaccine and 21.9% the second dose at the time of the study. Vaccine refusal was 0.6%, and 13.9% expressed some degree of vaccine hesitancy. Vaccine hesitancy and refusal was inversely associated with uptake of any dose of COVID-19 vaccine and their interest in protecting the family, coworkers, and their community. Conclusion: Agricultural workers in countries like Guatemala, have suffered a high incidence of asymptomatic and undetected SARS-CoV-2 infection. Most have received the COVID-19 vaccine, but there are moderate degrees of vaccine hesitancy that require better public health information to overcome it.