Fusarium wilt caused by Fusarium oxysporum f. sp. nicotianae (Johnson) W. C. Snyder and H. N. Hansen. is the most destructive disease of tobacco. The present study was carried out in vitro conditions to assess the possible use of biocontrol agents in field conditions. Trichoderma viride, Trichoderma harzianum, Bacillus subtilis and Pseudomonas fluorescens were evaluated for their antagonistic activity against F. oxysporum f. sp. nicotianae under in vitro conditions. Different biocontrol agents showed varying degrees of antagonism. T. viride and T. harzianum showed 70 and 40% inhibition of radial growth of the F. oxysporum f. sp. nicotianae respectively in dual culture technique. Mode of parasitism between the biocontrol agents and F. oxysporum f. sp. nicotianae were studied under the microscope. Formation of several loops and coiling of hyphae by T. viride and T. harzianum around the hyphae of the pathogen forming a thick compact rope like structure was observed. The biocontrol agents produced volatile metabolites. These metabolites suppressed the growth of F. oxysporum f. sp. nicotianae. The two biocontrol agents varied in the extent of volatile metabolite production. Among bacteria, B. subtilis inhibited the radial growth of F. oxysporum f. sp. nicotianae by 36%. The bacterial and fungal antagonists were not mutually antagonistic as the co-inoculation did not inhibit each other. The present work is the first report on the use of T. viride, T. harzianum and B. subtilis against F. oxysporum f. sp. nicotianae as potential biocontrol agents.