Abstract Background Therapeutic drug monitoring (TDM) of aminoglycosides and vancomycin is used to prevent oto- and nephrotoxicity in neonates. Analytical and nonanalytical factors potentially influence dosing recommendations. This study aimed to determine the impact of analytical variation (imprecision and bias) and nonanalytical factors (accuracy of drug administration time, use of non-trough concentrations, biological variation, and dosing errors) on neonatal antimicrobial dosing recommendations. Methods Published population pharmacokinetic models and the Australasian Neonatal Medicines Formulary were used to simulate antimicrobial concentration–time profiles in a virtual neonate population. Laboratory quality assurance data were used to quantify analytical variation in antimicrobial measurement methods used in clinical practice. Guideline-informed dosing recommendations based on drug concentrations were applied to compare the impact of analytical variation and nonanalytical factors on antimicrobial dosing. Results Analytical variation caused differences in subsequent guideline-informed dosing recommendations in 9.3−12.1% (amikacin), 16.2−19.0% (tobramycin), 12.2−45.8% (gentamicin), and 9.6−19.5% (vancomycin) of neonates. For vancomycin, inaccuracies in drug administration time (45.6%), use of non-trough concentrations (44.7%), within-subject biological variation (38.2%), and dosing errors (27.5%) were predicted to result in more dosing discrepancies than analytical variation (12.5%). Using current analytical performance specifications, tolerated dosing discrepancies would be up to 14.8% (aminoglycosides) and 23.7% (vancomycin). Conclusions Although analytical variation can influence neonatal antimicrobial dosing recommendations, nonanalytical factors are more influential. These result in substantial variation in subsequent dosing of antimicrobials, risking inadvertent under- or overexposure. Harmonization of measurement methods and improved patient management systems may reduce the impact of analytical and nonanalytical factors on neonatal antimicrobial dosing.