Adequate safety precautions and proper use of personal protective equipment (PPE) in hospitalized patients with health care-associated infections (HAIs) are the most effective preventions in their spread. Observational study was conducted in one inpatient facility in the Czech Republic before the COVID-19 pandemic (April 2019 to February 2020). Data were analyzed using Minitab at a significance level of .05. The execution of preventative measures in patients hospitalized with a HAI (n = 44) and the use of PPE by health care workers (n = 514) have been observed. The presence of defined PPE items usage differs on the basis of the department type (P = .0004). The correctness of PPE use differs on the basis of profession (P < .001), hand disinfection before PPE use (P < .001), use of all PPE (P < .001), and hand hygiene after PPE use (P < .001). General nurses had a higher observed frequency of hand hygiene (53.3%) than physicians (42.4%). The correct order of operations when using PPE differs on the basis of the department type (P < .001) and their architecture arrangement (open, semiopen, box) (P < .001). Critical preventative measures shortage was found at all units. Insufficient execution of the correct procedure of actions in the use of PPE was observed among health care workers. The best results have been observed among nurses.