This study was taken up to determine the prevalence of potential pathogens in most touched fomites in Neonatal Intensive Care Units (NICUs) and to determine their antibiogram. Fifty swabs each were collected from fomites from In-born (for babies born in our center) and out-born (for babies born elsewhere, brought to our center) NICUs, before and after cleaning. Samples were processed using standard Microbiological techniques. Antibiogram was determined by Kirby-Bauer disc diffusion method. From In-born NICU, before cleaning, 42 (84%) samples yielded growth with 48 bacterial isolates of which 31 (64.6%) were pathogens and after cleaning, 40 (80%) samples yielded growth with 41 bacterial isolates, of which 30 (73.1%) were pathogens. Most frequent isolates were non-fermenting gram-negative bacilli (NFGNB) and Klebsiella pneumoniae before and after cleaning, respectively. Prevalence of bacteria from In-born NICU before and after the cleaning was statistically significant (p = 0.025). From Out-born NICU, before cleaning, 42 (84%) samples yielded growth with 45 bacterial isolates, of which 25 (55.55%) were pathogens. Samples collected after cleaning; 42 (84%) samples yielded growth with 43 bacterial isolates of which 19 (44.18%) were pathogens. Most frequent isolates were NFGNB and Klebsiella pneumoniae from In-born NICU and K. pneumoniae and Escherichia coli from Out-born NICU before and after cleaning, respectively. Overall prevalence of pathogens was significant (p = 0.007). Staphylococcal isolates were resistant to Methicillin. NFGNB exhibited highest resistance (55.9%) towards Gentamicin. Significant decrease in the frequency of pathogens after cleaning indicates stringent practice of sterilization protocol would be essential in hospital environment.