The tracheostomized patients are colonized mostly by gram negative bacteria which lead to either tracheobronchitis or bronchopneumonia. This study was conducted to isolate and identify the potential pathogen causing post tracheostomy infection. A cross-sectional study was conducted during April 2008 to February 2009 based at Neuro Center, Kathmandu. Tracheal aspirates of 50 patients having fever more than 38°C were collected and analyzed for bacterial content. Out of the 50 cases, 45(90%) cases showed bacterial growth. Sixty-seven isolates were identified; with 20(44.4%) poly-microbial cases. Pseudomonas aeruginosa and enteric gram negative bacteria were predominant bacteria (n=27, 40.3%) followed by Staphylococcus aureus (n=7, 10.4%), other Gram negative bacteria (n=4, 5.9%) and Viridans Streptococci (n=2, 2.9%). Pseudomonas aeruginosa were most sensitive to the Amikacin (n=22, 81.4%) and Ciprofloxacin (n=19, 70.3%). All Pseudomonal isolates were resistant to the Cefotaxime. Enteric Gram Negative bacteria (EGNB) were most sensitive to Amikacin and Chloramphenicol (20, 74.0%) and all were resistant to Ampicillin and Cephalexin. All the gram positive bacteria isolated were sensitive to Vancomycin. Among the total isolates, 24 (88.8%) of Pseudomonas aeruginosa, 21 (66.6%) of enteric gram negative bacteria, and 5 (55.5%) of Gram positive bacteria were multidrug resistant (MDR). The study reported alarming condition of MDR in tracheal aspirates. So surveillance for source of multidrug resistant bacteria would be beneficial for intervention of infection related to it. Key words: Tracheal aspirates, polymicrobial growth, Multidrug resistant bacteria (MDR)10.3126/ijls.v4i0.3496International Journal of Life Sciences Vol.4 2010 pp.60-65