Butt rot disease, caused by Thielaviopsis paradoxa (De Seynes) Hohn., is one of the major diseases in pineapple cultivation in Malaysia. The objectives of this study were to evaluate the antifungal effect of antagonist bacteria against T. paradoxa, a causal agent of butt rot disease, and to observe the mechanism of antifungal activity of tested antagonist bacteria microscopically. In this study, in vitro antifungal potential of 5 antagonist bacteria, namely B1, B2, B3, B4, and B5, were isolated from infected and non-infected soil samples and evaluated using dual culture method against T. paradoxa. The mechanisms of antifungal activities of antagonist bacteria against the pathogen were microscopically observed. All of the bacteria showed inhibitory effects against the pathogenic fungi. B1 bacteria showed the highest inhibitory potential, with 73 % inhibition, followed by B2, B3, B4, and B5, with 71, 57, 56, and 48 % of inhibition compared to control, respectively. The results also showed that B2, B3, and B4 bacteria exhibited positive inhibition towards the pathogen, with more than 50 % percentage inhibition. The development of a new product for use as a biocontrol agent, used as an additional control or used in combination with existing ones, may reduce dependency on chemical control and increase antagonistic activity efficiency.