Healthcare-associated infections (HAIs) represent a major challenge in patient safety that affects services disproportionally. This paper aimed to assess how the HAI prevalence varies between hospital services and what contextual characteristics may explain such variance. A cross-sectional study was conducted on adult patients in Portuguese hospitals, using data from the European point prevalence survey of HAI prevalence. The study variables included patient, structural, and process variables, tested as risk factors, with patients clustered in hospitals. Variables with a p-value ≤ 0.2 in univariate analyses were retested in a multivariable model. A total of 18,261 patients from 119 hospitals were included: 736 from 56 intensive care units (ICUs), 3160 from 72 surgical departments, and 8081 from 90 medical departments. The HAI prevalence was 7.9%, 5.9%, and 1.7%, respectively. In ICUs, only the number of devices was associated with the HAI prevalence. In surgical departments, age, comorbidities, being a specialized hospital, and a higher ratio of infection prevention and control (IPC) personnel were associated with higher SSI. The safety climate was associated with lower SSI. In medical departments, age and devices were positively associated, whereas a larger ratio of IPC nurses was negatively associated. These results may help implement targeted interventions to achieve optimal results in each department.