Macrophomina phaseolina is a soil pathogen known as charcoal rot and can cause up to 90% yield loss in sunflower under suitable conditions. The serious damage caused by chemicals used in the control of soil-borne pathogens to the environment and health has become one of the most important concerns in agriculture. Therefore, in our study, it was aimed to determine the in vitro antagonistic effects of various bacterial species against M phaseolina. A total of 38 bacterial strains were isolated from soil samples in the rhizosphere of Malva sylvestris (hibiscus), Vicia sativa (vetch), Cicer arietinum (chickpea), Papaver rhoeas (weasel), Carlina marianum (thistle), Glebionis coronaria (crown daisy) and Vicia faba collected from Urla district of İzmir. All bacterial strains exhibited antibiosis effect under in vitro conditions, but it was determined that 5 bacterial isolates among them showed a high inhibition zone and showed an average inhibition potential ranging between 55% and 74%. The most effective bacteria identified at species and genus level by Maldi biotyping (MALDI-TOF MS) were identified as Bacillus amyloliquefaciens, Stenotrophomonas sp. and Bacillus cereus (3 isolates), and these species showed that they can be important biocontrol agents in biological control against M. phaseolina.