Background: Patients undergoing major open emergency abdominal surgery experience high morbidity and mortality rates and often have sepsis at admission. The purpose of this study was to evaluate the association between antibiotic timing and in-hospital outcomes such as complications, need for reoperation, length of stay, and 30-day mortality. Methods: This retrospective observational cohort study was conducted between January 2010 and December 2015 including patients that were triaged through the emergency department for subsequent major open abdominal surgery. All relevant perioperative data were extracted from medical records. The outcomes of interest were development of in-hospital postoperative complications, reoperations, length of stay, and 30-day mortality, all in association with antibiotic timing, categorized according to 0–6, 6–12, or > 12 h from triage. Multivariate logistic regression was performed to evaluate adjusted outcomes associated with antibiotic timing. Results: A total of 408 patients were included, of whom 107 (26.2%) underwent at least one reoperation and 55.4% had at least one postoperative complication. These complications consisted of 26% surgical complications and 74% medical complications. Of the surgical complications, 73% were Clavien–Dindo ≥ 3. The median length of stay was 9 days and the overall 30-day mortality was 17.9%. The data showed that the development of complications, need for reoperation, 30-day mortality, and the length of stay were significantly correlated to delayed antibiotic administration of more than 12 h from admission. Conclusions: Antibiotic administration more than 12 h from triage was associated with a significantly increased risk of postoperative complications, need for reoperation, 30-day mortality, and a prolonged length of stay, when compared to patients that received antibiotic treatment 0–6 h and 6–12 h after triage. Our data suggest that prophylactic antibiotics should be administered to all patients undergoing major open emergency abdominal surgery; however, the dose and duration cannot be concluded on the basis of our data and should be further examined.