We aimed to evaluate the clinical impact of shift patterns at work on gynecologic problems and the healthcare behavior of Korean nursing staff. We conducted a web-based survey for over two weeks in September 2021, involving female nursing staff, including nurses, nurses' aides, and nursing assistants, working in five medical centers. The questionnaire included 40 questions on baseline characteristics, working information, and experiences with gynecologic problems and medical approaches. Overall, 885 nursing staffs participated in the survey, of the 1,904 who received the invitation with an online link of the survey. The response rate was 46.5%. Among the participants responding to all questions, 569 (64.4%) worked two or three shifts and 305 (34.5%) worked full-time. In women rotating two or three-shift patterns, irregular menstrual cycles (21.6% vs. 13.8%, p = 0.005), abnormal menstrual cycles (40.9% vs. 33.8%, p = 0.038), and dysmenorrhea (48.0% vs. 38.4%, p = 0.006) were more frequent than in those who worked full-time. The experience of visiting gynecologic clinics (47.5% vs. 44.1%, p = 0.332) and treating gynecologic conditions (33.4% vs. 29.3%, p = 0.211) did not differ according to the working patterns. However, diagnosis of gynecologic diseases was more frequent in women working full-time (36.4% vs. 29.7%, p = 0.043). The rates of screening (76.1% vs. 57.8%, p<0.001) and human papillomavirus vaccination (55.7% vs. 39.9%, p<0.001) for cervical cancer prevention were higher in women working full-time than in two-three shifts. This study showed that rotating shift work may be related to an increase in gynecologic problems and a decrease in cancer prevention activity among female nursing staff.