Background: Patients in intensive care units (ICUs) are more prone to nosocomial infections caused by hospital strains of bacteria. These strains are often resistant to many antimicrobials. Objective: The aim of this study was to determine the bacterial profile and their drug sensitivity and resistance in different clinical specimens in ICUs of a tertiary care hospital. Materials and Methods: The study was conducted in different ICUs of a tertiary care teaching hospital in Western India, during April 2015 to March 2016. The clinical specimens received from ICUs were processed by standard method, bacteria were identified by VITEK 2 compact (biomerieux) automation system, and antimicrobial susceptibility testing was done with the same system to detect minimum inhibitory concentrations for penicillins, beta-lactam/beta-lactamase inhibitor, cephalosporins, carbapenems, aminoglycosides, tetracyclines, quinolones, folate inhibitors, nitrofurans, lipopeptides, and glycopeptides. Results: A total of 1849 clinical isolates identified were included in the study. Klebsiella spp. (n = 466) followed by Acinetobacter spp. (n = 377), Escherichia coli (n = 368), and Pseudomonas aeruginosa (n = 311) were among the maximum isolates. Most bacterial isolates (n = 1305) were from medical intensive care units. Maximum isolates were from endotracheal tube (n = 650). Colistin, tigecycline, minocycline, imipenem, and meropenem were the most common sensitive drugs for Gram-negative organisms. Conclusion: Optimum antimicrobial utilization in ICUs is important for better patient outcome and to prevent emergence of multidrug resistance. This can be achieved by strict infection control measures such as stringent adherence to hand washing practices, universal safety precautions, antibiotic policy formulation, and its implementation along with antibiotic stewardship program.