Background People experiencing homelessness (PEH) may be at increased risk of SARS-CoV-2 infection and severe COVID-19. The Dutch government established emergency shelters and introduced preventive measures for homelessness services and there were no major SARS-CoV-2 outbreak noticed among PEH during the first two waves of infections. This study aimed to assess the prevalence of current and past infections among PEH and staff at the end of the second COVID-19 wave by conducting an on-site COVID-19 screening project at homelessness services in Amsterdam, the Netherlands. Methods We assessed the proportion of visitors and staff members of four homelessness services at two locations in Amsterdam with positive SARS-CoV-2 qPCR and antibody results (IgG/IgM Rapid Test, Biozek) in May 2021. We also assessed sociodemographic, clinical and lifestyle characteristics, compliance with basic prevention measures and intention to vaccinate against COVID-19 among PEH and staff.Results A total of 138 visitors and 53 staff members filled out a questionnaire and were tested. Among PEH, the SARS-CoV-2 positivity rate was 0% (0/133;95%CI=0-1.9) and the antibody positivity rate was 1.6% (2/131;95%CI=0.8-7.5). Among staff, these percentages were 3% (1/32;95%CI=0.1-16.2) and 11% (5/53;95%CI=3.6-23.6), respectively. Most participants were frequently compliant with the basic preventive measures ‘not shaking hands’, ‘wearing a face mask’ and ‘washing hands’, but not with ‘physical distancing’. Overall, intention to vaccinate for COVID-19 was low (30% for visitors, 10% for staff) or medium (28% for visitors, 35% for staff). Trust in COVID-19 policies were also, for the most part, low (27% for visitors, 17% for staff) or neutral (32% for visitors, 53% for staff).Conclusions We observed a low prevalence of past and current SARS-CoV-2 infections among PEH, which may be explained by instated shelter policies, limited daily activities of PEH and compliance with certain prevention measures. Vaccine hesitancy and mistrust among visitors and staff could hinder vaccination uptake, suggesting that improvements in public heath communication and interventions towards visitors and staff of homelessness services are needed.