Abstract: This study was designed to evaluate the antagonistic effects of rhizobacterial antagonists against Coffee Wilt Disease (CWD) caused by Gibberella xylarioides under in vitro and in vivo conditions. Arabica coffee is Ethiopia’s main export crop. However, the production and productivity of coffee is being challenged primarily by coffee vascular disease (tracheomycosis).The greenhouse antagonism study was conducted with four antagonistic bacteria of one Bacillus (JU544) and three Pseudomonas spp. (JU941, JU13 and JU23). Out of 81 rhizobacterial antagonists tested on Half Strength King’s B (HSKB) medium against G. xylarioides, 13.6% of them significantly (p<0.0001) reduced the radial mycelial growth of the pathogen. From 11 rhizobacterial isolates tested for their phytobeneficial traits, eight of them produced protease. Nevertheless, five of them produced Hydrogen Cyanide (HCN) and other lytic enzymes. The bio-control agents, time of applications and the interaction of the two were significantly (p<0.0001) reduced the CWD severity and incidence under greenhouse conditions. The CWD control efficiency was significant (p<0.0001) and the highest bio-control efficiency was 72.64% when the coffee seedlings were treated with Bacillus spp. (JU544) seven days before the pathogen. The bacterial antagonists, time of applications and interaction of the two significantly (p<0.001) reduced the progression of CWD incidence. The rhizobacterial antagonists especially Bacillus spp. (JU544) effectively reduced CWD severity and incidence under greenhouse condition and can be further evaluated under field condition to ascertain their future applicability for inoculum development.