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Standard precautions (SPs) guidelines are the minimum infection prevention practices that apply to all types of patient care, regardless of suspected or confirmed infection status of the patient. They are based on risk assessment, make use of common sense practices and personal protective equipment that protect healthcare providers from infection and prevent the spread of infection from patient to patient. The aim of this study was to determine medical staff’s attitudes towards SPs and analyse the factors shaping these attitudes. The study was conducted using a questionnaire that comprised 25 statements describing the attitudes of medical personnel towards SPs. They were designed to pinpoint the factors that determine these attitudes. There were five factors identified that shape employees’ attitudes towards SPs: assessment of the situation, favourable patterns of behaviour, negative norms, unfavourable patterns of behaviour and rationalising. The study analysed 505 questionnaires filled in by hospital workers from five Polish cities. The majority of the respondents were women (92.1%), nurses (87.5%), the average age was 41.8 and the average seniority was 19.2 years. Over one-third of the respondents worked in non-surgical (36.4%) and surgical (31.6%) wards, 12.3% were employed in intensive care units (ICUs) and 8.9% in emergency departments (EDs). The variable significantly affecting the level of acceptance of SPs was seniority: initially the support was high, then it later decreased, with the greatest decrease occurring between the third and eighth year of work. The staff of medical wards and ICUs demonstrated significantly lower support for SPs and strong environmental impact on SPs perception, low degree of acceptance among medical ward staff correlated negatively with factors from the category “favourable patterns of behaviour”. The substantially strongest support for SPs was found in ED workers. The results indicate the need for continuous education of individual groups of workers concerning the application of SPs, but also the necessity to change the organisational culture in Polish hospitals.