Background: Varicella seroprevalence in healthcare workers at a tertiary care hospital in Taiwan was assessed following the inclusion of varicella zoster vaccination in the national vaccination schedule in 2004 and was made a hospital policy in 2008. Methods: Seroprevalence data were extracted from records of pre-employment health check-ups performed between 2008 and 2018 at a single medical center. Staff with complete medical records and anti-varicella zoster virus immunoglobulin G (VZV IgG) titers were included. Sex and age group differences in terms of geometric mean titer (GMT) were compared using analysis of variance and chi-squared tests. The significance of the correlation between age and the anti-VZV IgG titer was tested by linear regression. The odds of significant associations among age, sex, vocation, and the years of national and hospital adoption of vaccination were determined using univariate and multivariate analyses. p < 0.05 was considered statistically significant. Results: Of the 7314 eligible participants, 5625 (76.90%) were women, and the mean patient age was 26.80 ± 8.00 years. The lowest VZV-positivity rates were in 18–20-year-old women (85.16%; GMT, 362.89 mIU/mL) and men (87.59%; GMT, 288.07 mIU/mL). VZV positivity increased with age (p < 0.001). Participants born before 2002 were more likely to be seropositive than those born after 2003 (odds ratio, 2.51 vs. 1.0; p < 0.001). The lowest seropositive rate was found in the nursing staff (88.91%; 95% confidence interval, 87.74%–90.05%). Varicella vaccine boosters have been required at pre-employment health check-ups since 2008 if anti-VZV antibodies were not detectable. A follow-up evaluation found marginal significant differences in the odds ratios of seropositivity after 2007 (p = 0.052), especially in 2008 and 2014 (p < 0.05) after the hospital policy launched. Conclusions: Despite public health efforts, a small number of healthcare workers were inadequately protected, and antibody titers were lower than required to maintain herd immunity. For effective prevention of nosocomial infection, VZV IgG status should be documented for all HCWs, and susceptible HCWs should be vaccinated to avoid outbreaks. Pre-employment screening and vaccination have increased immunity and need to be conducted to ensure protection of vulnerable patients.