Background: Mumps, a contagious disease, is transmissible by respiratory droplet particles and is preventable by vaccination. In South Korea, mandatory vaccination against mumps has markedly reduced its incidence. However, both the incidence and the number of reported cases of mumps have persistently increased in South Korea since 2007. Despite high vaccination rates, mumps outbreaks continue to occur, and many studies have been conducted on mumps seroprevalence in children and adolescents. In comparison, few reports have been published regarding mumps seroprevalence in healthcare workers (HCWs) in South Korea. Objective: We investigated the seroprevalence of HCWs in South Korea. Methods: This study was conducted at Asan Medical Center, a 2,705-bed tertiary-care hospital in Seoul, South Korea, with 8,329 HCWs. In 2018, we performed mumps antibody testing for HCWs. We administered MMR vaccination to all HCWs whose antibody test yielded equivocal or negative results. However, we did not repeat mumps antibody testing after MMR vaccination. Results: In total, 6,055 HCWs (73%) underwent mumps antibody testing. The overall mumps seropositivity rate was 87% (95% CI, 86%–87%). Seropositivity rates of all birth cohorts ranged from 72% to 92%. Mumps seropositivity rates were 88% in HCWs born before 1970, 87% in those born between 1970 and 1989, and 88% in those born between 1990 and 1995 (P = .59). Mumps seropositivity rates for both women and men HCWs were 87% (3,770 of 4,311 women and 1,517 of 1,744 men); the difference was not statistically significant (P = .62). The overall mumps seropositivity rate was 87%, which was above the herd immunity threshold of 75%–86%. Conclusions: Our results revealed that the overall mumps seropositivity rate in South Korean HCWs was above the herd immunity threshold. On the basis of this finding, we recommend that MMR vaccination after serologic testing may be a more reasonable approach than universal MMR vaccination alone in Korea.