Background With the improvement of neonatal care in the country and survival of the preterm infants and sick neonates, many arising problems are being observed. One of these is the significant presence of neonatal thrombocytopenia and the need for lots of platelet transfusions per an infant. Many neonatal factors can cause thrombocytopenia, but we observed severe, prolonged thrombocytopenia in infants who received specifically vancomycin for sepsis or other medical/surgical conditions. Literature search revealed that vancomycin can cause immune thrombocytopenia by inducing platelet antibodies, though this is scarcely described in neonates. Participants and methods This is a hospital-based longitudinal study held in NICU during February 2017 to February 2019. All admitted neonates (term and preterm) who received vancomycin were involved, but those with maternal thrombocytopenia, systemic lupus erythematosus (SLE), maternal eclampsia/HELLP (Haemolysis, Elevated Liver enzymes, Low Platelets) syndrome, and Intr Uterine Growth Rrestriction (IUGR) were excluded. Results Of 117 infants admitted in this period, 68 infants fulfilled the inclusion criteria. The severe decline in platelet count observed on the second to third day of vancomycin treatment continued throughout the treatment and started to rise 2–3 days after discontinuation. During treatment with vancomycin, platelet transfusion 2–3 times a day was observed not to raise platelet level significantly, but it prevented serious bleeding. Conclusion Vancomycin-induced thrombocytopenia in neonate is a rising new problem in NICUs. The authors may need to add adjunctive intravenous immunoglobulins or methylprednisolone or change the dosing system to smaller frequent doses, given over longer time, to overcome this serious problem.