Plant tissue cultures can be contaminated by extensive diversity of bacteria and the contaminations are species specific. The contamination can reduce growth rate of shoot and root, multiplication factor, and even cause plant death. The study was conducted at Mekelle University, College of Veterinary Medicine and Tigray Biotechnology center and contaminated in vitro sugarcane culture obtained from laboratory of plant tissue culture. The objective of the study was to isolate, characterize, and identify bacteria from contaminated in vitro Sugarcane culture and to test the sensitivity of the isolates to the most commonly used antibiotics. Morphological, Gram stain, Endospore stain and Biochemical test method were used to isolate and identify the contaminates and disk diffusion method was used for the drug susceptibility test. Commonly available antibiotics were used for susceptibility testing. In the present study Escherichia, Bacillus and Micrococcus were isolated and identified as the major contaminant bacteria from in vitro sugarcane cultures. In antibacterial susceptibility test the isolates of Bacillus and Micrococcus were susceptible to Gentamicin, Chloramphenicol, Ciprofloxaciline, Tetracycline, Vancomycine, Streptomycin, Penicillin G and Kanamycine demonstrating the efficacy of these antimicrobials for the treatment of sugarcane in vitro culture contamination by incorporating in to the sugarcane media formulation while, the third isolate Escherichia were resistance to all antibiotic agents.