The emerging SARS-CoV-2 variants and waning vaccine-elicited immunity are two public health challenges that occurred simultaneously and synergistically during the summer of 2021 and led to a surging demand for COVID-19 vaccine booster dose (BD) rollout. This study aimed to evaluate the COVID-19 vaccine booster hesitancy (VBH) among Czech healthcare workers to explore the potential determinants of VBH. A national cross-sectional survey-based study was carried out between 3 and 11 November 2021, using an online self-administered questionnaire (SAQ) that explored the participants’ demographic characteristics, COVID-19 infection and vaccine anamneses, willingness to receive COVID-19 vaccine BD, and the psychosocial drivers of VBH. A total of 3454 HCW properly responded to the online SAQ, of which 80.9% were females, 30.3% were medical professionals, and 50.5% were ≤47 years old. Most of the participants were already inoculated against SARS-CoV-2 (95.2%), and BTN162b2 was the most commonly administered vaccine (90.7%). As the study sample was planned to represent the target population, it revealed a high level of BD acceptance (71.3%) among Czech HCW, while 12.2% were still hesitant and 16.6% were against the currently available BD. These results are consistent with other recent results from central Europe. Medical professional, male, and older participants were more likely to accept BD rather than allied health professional, female, and younger participants. The BDs’ perceived effectiveness against severe illness, symptomatic infection, and community transmission was a significant and strong predictor for BD acceptance, while the effectiveness against the circulating variants was not that important for our target population. The BDs’ perceived safety and ethical dilemmas of vaccine justice should be addressed sufficiently while communicating with HCW and other population groups. The altruistic reasons for BD acceptance, i.e., family protection, patient protection, and community health protection, underpin the recommendation of postponing the COVID-19 vaccine mandating in favour of stressing these altruistic concerns amid public health messaging.