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Abstract Background Increased workload and of the health workforce (HW) strained the capacity to maintain essential health services (EHS) during the Coronavirus Disease 2019 (COVID-19) pandemic, while putting them at increased risk of COVID-19 and other consequences to their health. The aim of this study was to assess the impact of COVID-19 on the health, wellbeing, and working conditions of nurses in Slovakia and to identify gaps in policies to be addressed to increase preparedness of the HW for future emergencies. Methods A nation-wide cross-sectional study was conducted among nurses during November–December 2021, referring to the period of January 2021 to November 2021. To assess the differences between impact on HW on various levels of care, respondents were grouped by type of facility: hospital-COVID-19 wards; Hospital–non-covid ward; Outpatient or ER; Other care facilities. Results 1170 nurses participated, about 1/3 of them tested positive for COVID-19 by November 2021, mostly developing mild disease. Almost 2/3 reported long-covid symptoms and about 13% reported that they do not plan to get vaccinated against COVID-19. The median of the score of the impact of workload on health was 2.8 (56% of the maximum 5), the median score of mental health-wellbeing was 1.9 (63% of a maximum of 3). The studied impacts in all domains were highest in nurses working in COVID-19 hospital wards. Significant disruptions of health care were reported, with relatively high use of telemedicine to mitigate them. Overall, about 70% of the respondents thought of leaving their job, mostly due to working stress or inadequate pay. Conclusions Our study showed that the COVID-19 pandemic poses a substantial burden on the health, wellbeing and working conditions of nurses in Slovakia and that a large proportion of nurses considered leaving their jobs because of work overload or low salaries. Human resource strategies should be adopted to attract, retain and continuously invest in HW development including in emergency preparedness and response. Such an approach may improve the resilience and preparedness of the health system in Slovakia for future emergencies.