The article's subject identifies employee groups affected by the implementation of Industry 4.0 in business practice, either positively or negatively. Apart from the identification of the groups in question, it is essential to name where the disadvantage lies from the subjective point of view of the employees but also the objective point of view. The implementation of Industry 4.0 in enterprises brings not only a threat in the form of redundancy of the current workforce but also an opportunity to make the employees' work performance more efficient and improve their working conditions. Several analytical methods were used to obtain comprehensive analytical background. They were used to identify disadvantaged groups of employees, describe the groups of employees and to what extent they are affected by the work activity, and where their disadvantage lies in implementing Industry 4.0. A comprehensive assessment of the identified disadvantaged groups of employees within Industry 4.0 serves to understand the threats to the workforce better and allows for the targeted implementation of policies and practices to work with the current crew in the changing ways of working within Industry 4.0 and thus also contribute to the prevention of work-related diseases and workforce devaluation.