BACKGROUND: Controlling antibiotic resistance of bacteria is a priority for public healthcare. AIM: This study concerned the frequency of multidrug resistant bacteria (MDRB) in a Tunisian Hospital with the aim of establishing guidelines for MDRB prevention. METHODS: The study was conducted during two years (1 January 2005-31 December 2006). Samples collected for the clinical diagnostic were included. The MDRB concerned were: methicillin resistant Staphylococcus aureus(MRSA), Enterobacteriacae resistant to of third generation cephalosporin (ER3GC), Acinetobacter baumannii resistant to both imipenem and ceftazidime, Pseudomonas aeruginosa resistant to both imipenem and ceftazidime. RESULTS: During the study period, 2475 bacteria were tested by disk diffusion. 597 MDRB were collected, the rate of MBR was 24.1%. These MDRB were mainly recovered in burn unit (82.6%). ER3GC (47%) and MRSA (29,2%) were the most frequent MDRB. A. baumannii and P. aeruginosa multiresistant concerned 14,8% and 9% of MDRB. MDRB were isolated mainly from blood cultures (45%). The rate of MRSA was 46.4% among 375 strains of S. aureus. ER3GC represented 25,6% among 1096 isolates. Concerning A. baumannii and P. aeruginosa, 51.7% and 20.5% were resistant to both imipenem and ceftazidime among 170 and 264 isolates. Antibiotic resistance evolution showed a decrease of resistance in 2006 versus 2005. This decrease should be explained by the improvement of hygiene measure especially hand washing with the introduction of hydro- alcoholic solutions, a better targeted antibiotherapy promoted by a close cooperation between microbiologists and clinicians. CONCLUSION: The MDRB were frequent in our hospital. They were mainly isolated from the burn department. The measures of prevention already implemented are effective and must be strengthened with the continuous surveillance of MDRB.