Introduction: Although hand hygiene and use of gloves as protective measures against pathogen transmission are indisputable, the level of compliance with the worldwide-accepted hygiene procedures remains below the expectations. Work overload has been specified as one of numerous barriers limiting compliance with the directives. The aim of the study was to assess the impact of actual workload on compliance with the recommendations on hand hygiene and use of protective gloves by hospital medical personnel, as well as to recognize subjective opinions regarding the workload impact on compliance with hygiene procedures. Material and methods: The study employed a quasi-participation method. It comprised all members of medical personnel (186 nurses and physicians) working at six wards of three hospitals of the Lodz region. A total of 1544 observation units were surveyed, with a total of 4101 hygiene-requiring activities. The workload was defined as an activity index and an effective workload index. An additional questionnaire survey was carried out in order to identify subjectively-perceived impediments in the application of hygiene recommendations. Results: Work overload has been specified as one of numerous barriers limiting compliance with the directives. The study confirmed the work overload impact on ignoring the hygiene recommendations. Medical personnel on night calls more seldom comply with hand hygiene than during the morning and day calls. The majority of medical personnel stated that reduced workload and more efficient work organization should contribute to the improvement of hygiene-related behaviors of medical personnel. Conclusions: Higher work overload may increase the frequency of non-compliance with hand hygiene. There is a necessity for continuous education and improvement of professional qualifications of medical staff regarding hospital hygiene.