Evidence implicates vitamin D deficiency in poorer outcomes and increased susceptibility to hospital-acquired infections (HAIs). This study examined the association between serum vitamin D levels and HAIs in a population of hepatobiliary surgery patients. Participants in this prospective analytical observational study were patients who underwent hepatobiliary surgery in a tertiary hospital in Aragon, Spain, between February 2018 and March 2019. Vitamin D concentrations were measured at admission and all nosocomial infections during hospitalization and after discharge were recorded. The mean 25-hydroxyvitamin D concentration of the study population (n = 301) was 38.56 nmol/L, which corresponds to vitamin D deficiency. Higher vitamin D concentrations were associated with a decreased likelihood of developing a HAI in general (p = 0.014), and in particularly surgical site infection (p = 0.026). The risk of HAI decreased by 34% with each 26.2-nmol/L increase in serum vitamin D levels. Vitamin D levels may constitute a modifiable risk factor for postoperative nosocomial infections in hepatobiliary surgery patients.