Abstract Optimizing sepsis proficiency of healthcare professionals is key to improving outcomes. We aimed to evaluate sepsis knowledge and management proficiency in neonatal units in Switzerland. This cross-sectional survey, tailored to profession, probed physician and nurses’ self-assessment of sepsis knowledge and management proficiency, sepsis definitions and epidemiology, and sepsis diagnostics and management proficiency in seven of nine tertiary care neonatal units. For proficiency, points were given based on the accuracy of answers on diagnosis and management in vignette clinical questions. Participants’ scores were computed by summing points and expressed in percent of the total available points. The questionnaire was completed by 108/158 physicians (68.3%) and 418/732 nurses (57.1%). Median proficiency of physicians and nurses were 73.1% (Q1-Q3, 68.8–77.4%) and 59.1% (Q1-Q3, 52.3–65.9%). Physicians displayed an imbalance between proficiency and self-assessment. Nurses displayed a significant association between proficiency and self assessment of management but none with self-assessment of knowledge. A higher proficiency in theoretical questions did not correlate with proficiency in decision-making (physicians: R = 0.13, nurses R = 0.24). For instance, while over 90% of physicians and 80% of nurses recognized clinical risk factors and signs of sepsis, and actions to prioritize, in a vignette, 22.2% of physicians and 34.0% of nurses did not recognize the need for antibiotics within 1 h in an extremely preterm newborn with signs of deterioration and phlebitis around a central line. We identified knowledge gaps, a need to better transduce knowledge into decision-making, and potential for improved self-assessment. Targeted simulation training could address these shortcomings.