Objective The new coronavirus disease 2019 (COVID-19) is a major health problem worldwide . The surveillance of seropositive individuals serves as an indicator to the extent of infection spread and provides an estimation of herd immunity status among population . Reports from different countries investigated this issue among healthcare workers (HCWs) who are “ at risk ” and “ sources of risk ” for COVID-19 . This study aims to investigate the seroprevalence of COVID-19 among HCWs in one of the COVID-19 referral centers in Makkah, Saudi Arabia using three different serological methods . Methods In-house developed enzyme-linked immunoassay (ELISA), commercially available electro-chemiluminescence immunoassay (ECLIA), and microneutralization (MN) assay were utilized to determine the seroprevalence rate among the study population . 204 HCWs participated in the study . Both physicians and nurses working in the COVID-19 and non COVID-19 areas were included . Twelve out of 204 were confirmed cases of COVID-19 with variable disease severity . Samples from recovered HCWs were collected four weeks post diagnosis . Results The overall seroprevalence rate was 6.3% (13 out of 204) using the in-house ELISA and MN assay and it was 5.8% (12 out of 204) using the commercial ECLIA . Among HCWs undiagnosed with COVID-19, the seroprevalence was 2% (4 out 192). Notably, neutralizing antibodies were not detected in 3 (25 %) out 12 confirmed cases of COVID-19 . Conclusions Our study, similar to the recent national multi-center study, showed a low seroprevalence of SARS-Cov-2 antibodies among HCWs . Concordance of results between the commercial electro-chemiluminescence immunoassay (ECLIA), in-house ELISA and MN assay was observed . The in-house ELISA is a promising tool for the serological diagnosis of SARS-CoV-2 infection . However, seroprevalence studies may underestimate the extent of COVID-19 infection as some cases with mild disease did not have detectable antibody responses.