Background: Attitude is a relatively permanent inclination towards a positive or negative evaluation of a given social or physical object, which determines a person’s disposition towards their surrounding social reality and informs his/her behavior. Aims: The aim of this study is to assess the attitudes of nursing staff, in terms of the emotional and behavioral components, in relation to selected social groups: a Roma person, a hearing-impaired person, a Muslim, and a person of a homosexual orientation. Design: This cross-sectional study was conducted by means of an Internet questionnaire. Methods: This study included 3900 nurses from Poland who were participants in social networking sites and discussion groups for nurses. The study data were collected by using a self-constructed survey questionnaire. The results were reported using the STROBE Checklist. Results: The following scale was adopted: mean 1.0–3.5—positive attitude, 3.6–6.0—negative attitude. Respondents showed positive attitudes towards patient groups (1.67–2.30), the least positive being towards Muslims (2.30) and Roma (2.21). The respondents predicted that during the performance of professional activities, they would have the biggest problem with a person of homosexual orientation (22.1%) or a Muslim person (19.0%). The results show that the age and length of service most often influence attitudes towards patients from different social groups. Conclusions: Respondents with a longer period of work experience and respondents with lower education, despite declaring positive attitudes towards the surveyed social groups, expressed negative statements towards Muslims and homosexuals. Cultural education during the undergraduate and postgraduate studies of nursing staff is essential. Impact Statement: This research indicates that the lesser the need for direct involvement in interactions with patients from other groups, the greater the willingness to accept the situation in which care is provided.