Anidulafungin, an echinocandin, is currently approved for treatment of fungal infections in adults. There is a high unmet medical need for treatment of fungal infections in neonatal patients, who may be at higher risk of infections involving bone, brain, and heart tissues. This in vivo preclinical study investigated anidulafungin distribution in plasma, bone, brain, and heart tissues in neonatal rats. Postnatal day (PND) 4 and PND 8 Fischer (F344/DuCrl) rats were dosed subcutaneously once with anidulafungin (10 mg/kg) or once daily for 5 days (PND 4–8). Plasma and tissue samples were collected and anidulafungin levels were measured by liquid chromatography-tandem mass spectrometry. The mean plasma Cmax and AUC0-24 values were consistent with single-dose plasma pharmacokinetics (dose normalized) reported previously for adult rats. Observed bone concentrations were similar to plasma concentrations regardless of dosing duration, with bone-to-plasma concentration ratios of approximately 1.0. Heart concentrations were higher than plasma, with heart to plasma concentration ratios of 1.3- to 1.8-fold. Brain concentrations were low after single dose, with brain-to-plasma concentration ratio of approximately 0.23, but increased to approximately 0.71 after 5 days of dosing. Tissue concentrations were nearly identical after single-dose administration in both PND 4 and PND 8 animals, indicating that anidulafungin does not appear to differentially distribute in this period in neonatal rats. In conclusion, anidulafungin distributes to bone, brain, and heart tissues of neonatal rats; such results are supportive of further investigation of efficacy against infections involving bone, brain, and heart tissues.