Objective To understand the contact behavior between the hands of caregivers of patients with multidrug-resistant organism (MDRO) infection, environmental surfaces and caregiver themselves. Methods Caregivers of MDRO-infected patients in two university affiliated hospitals were directly observed from March 2019 to July 2020, self-designed observation scale was used by trained observers to record the contact situation and frequency of caregivers' hands with patients, environmental surfaces and caregiver themselves. Results Patients were most frequently contacted by caregivers (89.82%), followed by areas adjacent to patients (88.94%). The frequently contacted environmental surface by caregivers included bed-rail (80.09%) and bed surface (68.58%). 46.02% and 36.73% of the caregivers touched the mask and trunk during self-contact respectively. The total contact frequency between caregivers' hands and environmental surface was different in different departments, and the contact frequency of internal medicine was higher than that of surgery (P < 0.05). The contact frequency between caregivers' hands and patients was different in different nursing periods, contact frequency with patients in peak period of treatment and nursing as well as visit period was higher than that in morning and evening nursing period (P < 0.05). Conclusion When caregivers provide care for MDRO-infected patients, frequent contact with patients, environmental surface and caregiver themselves may lead to the spread of MDRO infection.