Background: The frequent use of invasive procedures in intensive care units (ICUs), such as tracheostomy, is associated with increased risk of nosocomial infections of the lower respiratory tract with increase in incidence of antibiotic resistant bacteria. There is inadequate published information regarding the lower respiratory tract bacterial pathogens and their resistance patterns in Indian clinical setting. Hence we endeavor to determine the pattern of colonization of tracheal aspirate and their antimicrobial sensitivity. Materials and methods: The study was conducted in the Department of Otorhinolaryngology at a Tertiary Care Center at Belagavi from January 2012 to December 2012. The tracheal aspirate was collected intraoperatively on day 1 and on day 8 during tube change using 10F suction catheter under direct vision. The sample was immediately transferred to fluid Thioglycollate medium and transported to the Microbiology laboratory. The aerobic culture and sensitivity and anaerobic culture were done. Results: In this study, on day 1, 26 (87%) patients showed growth in tracheal aspirate as compared to 28 (93%) on day 8 following tracheostomy. The most common organism isolated in day 1 and day 8 aspirate was Pseudomonas aeruginosa which was present in 11 (40%) and 13 (45%) of isolates, respectively. The antimicrobial sensitivity of the organisms in the aspirate decreased as the duration of tracheostomy increased probably due to the development of hospital-acquired resistance and/or infection. Anaerobic organisms were also isolated; however, they were insignificant. Conclusion: The observation of the varying pattern of the organisms and antimicrobial susceptibility will definitely prove fruitful in the treatment of infections of the lower respiratory tract under our clinical settings. The regular surveillance culture of aspirates is of prime importance in patients suspected of having infection such as tracheobronchitis and pneumonia.