Introduction: The rate and extent of neonatal gastrointestinal tract colonization depends on various perinatal and neonatal factors. Colonization of gastrointestinal tract (GIT) after birth could be delayed because of prematurity, caesarean delivery or total parental nutrition. The ill and preterm neonates in NICU acquire intestinal flora that differ from that of healthy newborn. Objective: Hence we aim to study the factors associated with pathological colonization of aerobic microbial flora in neonates and find pattern of pathogenic aerobic microbial colonization of neonates. Methodology: The study was done on 94 neonates in a tertiary teaching hospital NICU , 51 cases with pathological colonization ,43 control with normal colonization. Data collected using Performa, Oral and rectal swabs taken Results: 68 neonates who received antibiotics 46 (90.2%) were cases 22(51.3%) were controls. Staphylococcus aureus was predominant suggesting influence of maternal colonization. Cases were more in babies born through LSCS and in babies whose mother had received antibiotics Conclusion: Of all factors influencing establishment of pathological microbiota in neonatal GIT which are interdependent , neonatal exposure to antibiotics was the most significant . Emergence of MRSA , ESBL are of particular concern as these are a constant threat as source of health care associated infection.