Background: Patients at intensive care units (ICUs) are vulnerable to acquiring nosocomial infections. The main objective of this study was to explore and characterize the burden of nosocomial infections from an ICU of National Medical College and Teaching Hospital (NMCTH), Birgunj, Nepal. Methods: A prospective hospital-based study was conducted between April and December 2018 at NMCTH, Birgunj, Province 2, of Nepal. A total of 374 specimens including clinical specimens ( n = 190) from patients admitted in an ICU and animate and inanimate environmental samples ( n = 184) from the ICU were collected. Collected specimens were cultured in specific microbiological media, and microbial isolates were identified and subjected to antibiotic susceptibility test. Results: Altogether, 374 specimens (190 clinical specimens and 184 nonclinical) of an ICU were analyzed. Out of 190 clinical specimens, 51% (97/190) showed bacterial growth. Isolated bacteria were Staphylococcus aureus (33%; 32/97), Escherichia coli (20.6%; 20/97), Klebsiella spp. (15.5%; 15/97), Pseudomonas spp. (11.3%; 11/97), and Acinetobacter spp. (11.3%; 11/97). Out of 184 nonclinical specimens, 51.6% (95/184) of the samples showed microbial growth. Among the isolates, Klebsiella spp. predominated (30.6%; 26/85) the growth, followed by S. aureus (22.4%; 19/85), Acinetobacter spp. (21.2%; 18/85), and Pseudomonas spp. (17.6%; 15/85). Among all clinical and nonclinical isolates, 61.9% (60/97) of the clinical specimens and 65.9% (56/85) of the nonclinical specimens showed multidrug resistance (MDR). Conclusion: Two-thirds of the specimens from both clinical and nonclinical specimens showed MDR. Urgent actions are required to address the augmented rate of nosocomial infections and MDR bacteria among ICUs in Nepal.