Background: Sepsis is defined as life-threatening organ dysfunction caused by an unregulated host response to infection. The emphasis is on the imbalance of homeostasis and the response to infection, as well as mortality and the importance of recognizing sepsis as early as possible. The knowledge of undergraduate nursing students is an extremely important indicator for future work in the healthcare system after graduation. The aim of this study was to investigate the levels of knowledge about sepsis among undergraduate nursing students and to compare differences in different years of study, as well as differences in their study model. Methods: A cross-sectional study was conducted on 618 nursing students at the University of Applied Health Sciences in Zagreb, Croatia. All three years of study and both full-time and part-time (employed) nursing students were included. The questionnaire “Determinants of Sepsis Knowledge” was used in the research. Results: The percentage and number of third-year students who correctly answered the items on Knowledge of Sepsis were statistically significant compared to the first two years of study. The percentage and number of employed students who responded correctly to the items on Knowledge of Sepsis were statistically significant compared to students who were not employed. Conclusions: The ability of nursing students to recognize and respond to the deterioration in a patient’s condition due to sepsis is very important, so appropriate education about sepsis is essential. We recommend a greater representation of sepsis content in the core curriculum of nursing students’ education in terms of theoretical instruction and clinical and simulation exercises.