The possibility of employing antagonistic bacteria for control of tobacco brown spot was studied. Approximately 136 strains of bacteria were isolated from phyllospheres of tobacco and 9 of these possessed high levels of antagonistic properties. They significantly reduced brown spot in detached tobacco leaves when artificially inoculated with Alternaria alternata . Culture filtrate of the most effective bacterial isolate which designated as Tpb88 was shown to be very efficient in inhibiting mycelial growth of A. alternata in dual cultures. Culture filtrate of Tpb88 inhibited germination and germ tube elongation of A. alternata . The results show that the culture filtrate directly inhibited spore germination of A. alternata , especially during the first hours of the paired cultivation. The rate of antagonistic activity of culture filtrate of Tpb88 depended on its concentration in the mixture. The greatest inhibition of spore germination was observed at the highest concentration of filtrate (filtrate to fungal spores inocula mixed in proportion 1:50). These suggested that the hypothetic mechanism of Tpb88 against tobacco brown spot was producing antagonistic substances.