Background: Strong patient safety culture and effective teamwork are necessary for patient safety. Registered nurses (RNs) and nursing assistants (NAs) work closely with patients, and effective teamwork and communication skills are essential.Aim: To describe and compare the perceptions of patient safety culture, teamwork, and attitudes toward teamwork among RNs and NAs in hospital wards (medical and intensive care), and to explore the potential associations between teamwork and patient safety culture.Methods: A cross-sectional study was conducted in two hospitals in a single region of Sweden. In one hospital, RNs (n = 103) and NAs (n = 59) in medical care, and RNs (n = 41) and NAs (n = 28) in intensive care were invited to participate. In the other hospital, RNs (n = 67) and NAs (n = 66) involved in medical care were invited. The survey included the Swedish version of Hospital Survey on Patient Safety Culture (S-HSOPS), the TeamSTEPPS Teamwork Perceptions Questionnaire (T-TPQ), and the TeamSTEPPS Teamwork Attitudes Questionnaire (T-TAQ).Results: In total, 155 nurses responded to the survey (response rate = 43%). Overall and in medical care, NAs had a significantly higher score than RNs in "handoffs and transitions" and in intensive care in "information and support to patients and family who have suffered an adverse event" (S-HSOPS). RNs and NAs in intensive care reported higher scores in "communication" (T-TPQ) than RNs and NAs in medical care. Overall and in medical care, RNs had more positive attitudes than NAs in total T-TAQ, "mutual support," and "communication." T-TPQ were associated with "overall perceptions of patient safety" and T-TAQ were associated with "frequency of error reported."Conclusion: This study shows differences between RNs and NAs working in medical and intensive care in their perceptions of patient safety culture, teamwork, and attitudes toward teamwork. Teamwork perceptions are important for overall patient safety.