Aims: Temporary Immersion Bioreactor (TIB) system is an advanced technology for commercial mass production of potato microtubers. Despite of several advantages, this system possess a great risk of culture loss at any stage of micropropagation due to microbial contamination. The aims of this study were to identify microbial contaminants isolated during potato shoot growth in the TIB system, evaluate the efficacy of antimicrobial agents to prevent them, to investigate the effect of those agents in vitro on growth and morphology of potato plantlets. Methodology and results: Six bacteria namely Pseudomonas, Staphylococcus, Klebsiella, Corynebacterium, Proteus, Bacillus and five fungi Aspergillus, Penicillium, Mucor, Fusarium and Rhizopus were isolated from the TIB system. We examined the effect of three antibacterial (Gentamycin, Vancomycin and Tetracycline) and four antifungal agents (Mencozeb, Propiconazole, Bavistin and Copper oxychloride) on the contaminants and on potato shoot growth. Results show that Gentamycin (50 mg/L) and Propiconazole (0.15%) were most effective against the isolated bacteria (35 mm inhibition zone) and fungi (100%) respectively, whereas Gentamycin in combination with Bavistin showed better performance on potato shoot and root development. Conclusion, significance and impact of study: Present study will provide useful guidelines to reduce or eliminate the risk of contamination during micropropagation.