Tracheostomy is a commonly performed airway surgery for critically ill patients. Tracheostomy tube is an indwelling prosthesis, providing potential surface for growth of bacteria. Biofilm formation by bacteria as a self-protective mechanism, has led to worrisome antibacterial resistance and thus higher rate of nosocomial infections. A prospective observational study was conducted with a purpose of knowing most common organisms capable of forming biofilm on tracheostomy tube and their antibiotic sensitivity in our setting. Fifty seven percent of the isolates were found to be capable of biofilm production. Acinetobacter baumannii (45%) was the commonest biofilm producer isolated and the commonest multidrug resistant organism (35.7%), followed by Klebsiella pneumoniae (28.5%) . Both biofilm producers and non-biofilm producers were found most susceptible to Amikacin (43%), followed by Gentamicin (30%) and Ciprofloxacin (18.5%). No significant association was found between biofilms and ventilators (p value = 0.558) or pre-existing infection (p value = 0.66) using Chi square test. Potentially biofilm producing bacteria were isolated from tracheostomy tube inner surfaces just after a week of their insertion, in majority of patients. Acinetobacter baumannii and Klebsiella pneumoniae were the commonest biofilm forming organisms and Amikacin, Gentamicin and Ciprofloxacin were most sensitive drugs. Multi drug resistant organisms were also commonly found, stressing the need for sensitivity-based antibiotics. Ventilator usage had no strong association with biofilm formation. Patients with non-infectious conditions also harboured bacteria capable of biofilms in tracheostomy tubes demanding the need for stringent tube hygiene measures and prophylactic antibiotics.