Antimicrobials are frequently prescribed to neonates who require hospital care, but the influences on clinical decision-making and practice variation in this process are ill-understood. We performed a cross-sectional survey of practitioners who prescribe antimicrobials in 3 Australian neonatal units.During two 5-day data capture periods per center, 56 practitioners reported their general confidence in antimicrobial decision-making for neonates. Then, 4 questionnaires evaluated diagnostic certainty and influences on antimicrobial decision-making for 68 antimicrobial courses and 11 infection evaluations where antimicrobials were not prescribed.Self-reported guideline use at antimicrobial commencement was high (26/31, 84%). Clinical risk factors, clinical signs and laboratory tests contributed variably to decisions to start and cease antimicrobials. Consultation with a colleague contributed to 14/31 (45%) decisions to commence antimicrobials and 13/34 (38%) decisions to cease them. The most frequent responses to questions regarding the likelihood of infection and the possibility of an alternative diagnosis were "some possibility" and "some likelihood." Team concordance in responses ranged from 14% to 50%. While practitioners in roles that denoted more clinical experience had greater general confidence in antimicrobial decision-making, this difference was not observed in real-world clinical situations where infection was not microbiologically confirmed.Clinical, laboratory, practitioner, team and center-based factors each influence antimicrobial prescribing decisions. Clinical uncertainty and differing guidelines likely contribute to practice variation. Future work to inform stewardship efforts should include improved guideline consistency, roles of diagnostic aids and a better understanding of the medicocultural contributors to neonatal antimicrobial prescribing.