Neonates admitted to neonatal intensive care units are at a risk of developing healthcare-associated infections, leading to increased risk of mortality. This study aimed to identify organisms causing such late-onset infections in neonates and determine whether these isolates were genetically identical to those from the surrounding environmental surfaces and hands of healthcare workers (HCWs). A cross-sectional study was carried out over a period of 4 months in a university neonatal intensive care unit (NICU). Samples were collected from all neonates with symptoms of late-onset infections (n = 180). Fingerprint samples of 21 healthcare workers as well as 330 random environmental samples were also taken from the unit. Isolates from neonates, environment and fingerprints were subjected to protein electrophoresis followed by sequencing to detect genetic similarities. Almost half of neonatal samples were culture positive (91/180, 50.6%), out of which 72% of bacterial isolates (49/68) were multi-drug resistant. Klebsiella pneumoniae (32.6%) and Candida spp. (28.4%) were the commonest neonatal isolates. A cluster of two homologous Klebsiella pneumoniae strains was isolated from a neonate and an examining bed, while another homologous cluster was from a neonatal sample and a portal incubator. A third cluster was isolated from hands and three neonatal samples. This cluster (caused by Klebsiella pneumoniae strain NH54 chromosome) was found to perpetuate over the 4 months of the study. All three clusters were multi-drug-resistant Klebsiella pneumoniae. A homologous pair of each of Candida tropicalis and Candida glabrata was isolated from the blood of two neonates, and one neonatal and a crash cart sample, respectively. Overall, 8.8% (8/91) of neonatal samples were found to be homologous to other neonatal/environmental/hand isolates, denoting perpetuation of pathogens between neonates themselves and also other reservoirs of infections.Conclusion: The hands of HCWs, crash carts and incubators are reservoirs of pathogens and can lead to nosocomial infections. Clusters of multi-drug-resistant Klebsiella pneumoniae and Candida spp. were the predominant neonatal pathogens in this NICU. What is Known: • The role of hands and the environment in transmission of infections to neonates is a subject of debate. • Genetic sequencing provides solid evidence for detecting homologous strains. What is New: • K. pneumoniae was the most frequently isolated pathogen, and concomitant isolation was found in two cases from the neonatal surroundings (bed/incubator) and hands. • Candida spp. with homology were also found in different neonates and environmental samples suggesting risk of transmission.