The true burden of COVID-19 in Yemen is underestimated. The healthcare system is dysfunctional and there is a high shortage of health care workers in the country. Testing for SARS-CoV-2 remains limited and official surveillance data is restricted to those who are severe or highly suspected. In this study, Médecins Sans Frontières (MSF) aimed to conduct serological screening using rapid tests for asymptomatic staff at the MSF Aden Trauma Center to determine the SARS-CoV-2 antibody seropositivity. Four months after the peak of the first wave, we offered all the staff at the MSF Aden Trauma Center PCR if symptomatic, and a baseline SARS-CoV-2 serology screening followed by follow-up screenings. A final round was scheduled four months after the baseline. A rapid serology lateral flow test, NG-Test IgM-IgG was used in all rounds and in the final round, an electrochemiluminescence immunoassay (ECLIA) (Elecsys Anti-SARS-CoV-2 assay). Univariate and multivariate analyses were used to identify risk factors for seropositivity. The level of agreement between the different serology assays used was investigated. Overall 69 out of 356 participants (19.4%, 95% CI 17.9–20.8) tested positive by NG-Test between September and November 2020. A sub-sample of 161 staff members were retested in January 2021. Of these, the NG-Test detected only 13 positive cases, whereas the ECLIA detected 109 positive cases. The adjusted seroprevalence by ECLIA was 59% (95%CI 52.2–65.9). The non-medical staff had significantly lower odds of seropositivity compared to the medical staff (AOR 0.43, 95% CI 0.15–0.7, p<0.001). The positive percent agreement between the two tests was very low (11%). Our results suggest a very high SARS-CoV-2 seroprevalence in healthcare workers in Yemen, highlighting the need for regular testing and rapid vaccination of all healthcare workers in the country.