Background: Cleaning workers face many workplace risk factors and may experience many health problems. In this context, this study aimed to evaluate the musculoskeletal disorders, the health status of hospital cleaning workers, and the working conditions and risk factors affecting the workplace environment. Material and Methods: This cross-sectional study was conducted in a university hospital of Turkey. All the cleaning workers who have been working for ≥1 year were participants. The participants’ socio-demographic and occupational characteristics, health complaints, workplace risk factors, occupational accidents, and ergonomic nonconformities were observed and questioned. Results: Four hundred thirty-eight cleaning employees participated in the study. In the past year, 19.6% of participants had an occupational accident. Of those, 24.4% did not report it, and 30.2% were absent from work. No pre-employment examination was reported by 36.8% of the participants, and periodic medical examinations were never undergone by 98.4%. Low back pain was experienced by 42.0% of the participants, while 29.5% reported shoulder pain and 28.8% knee pain. While working, 83.1% of the participants bent frequently, 82.2% repeated the same movement, and 73.2% stood for a long time. Chemical substances were the most common workplace risk factors. There were significant differences according to age and gender in almost all musculoskeletal disorders. Gender differences were observed also in various health outcomes and occupational complaints. Repeated bending and prolonged standing were associated with hip/leg and foot/ankle pains; heavy lifting with low back, back, wrist/hand, and ankle/foot pains; and failure to the breaks with shoulder, knee and hip/leg pains. Conclusions: This research investigated the health issues and occupational safety challenges faced by hospital cleaning personnel. Specifically, it examined musculoskeletal disorders and work-related accidents, emphasizing gaps in regular health screenings for these workers. The findings underscore gender variations in these challenges and propose strategies to mitigate ergonomic risks encountered by cleaning staff. Med Pr Work Health Saf. 2024;75(5):397–413