Background Blackleg and tuber soft rot are among the most important potato diseases caused by the bacteria belonging to the genera Pectobacterium. This pathogen causes significant economic losses each year. The antagonistic activity of different bacterial cultures against this pathogen was studied. Results Six hundred eight bacterial cultures isolated from potato tubers and rhizosphere soils procured from different locations across Morocco were tested for their antagonistic activity against Pectobacterium carotovorum. Forty isolates, all originating from tubers, showed positive antagonistic activity during preliminary screening. Among the 40 isolates, 10 were found to have a symptom suppression superior to 90%. Of the 10 isolates, 9 showed clear zone in the agar medium (in vitro test), with differences between antagonist’s inhibition diameter. For the in vivo test, 8 isolates induced total suppression of soft rot on potato slices (in vivo test). The other 2 biocontrol strains (Amo-23 and Atd-2) were capable to minimize soft rot symptoms of up to 94.4 and 96.2%, respectively. Among the selected strains for in planta experiment, 6 strains (namely Ame-4, Atd-2, Atd-4, Ag-216, Al-51, and Ama-501) showed total reduction of disease symptoms. Biochemical and molecular tests identified 8 strains of Bacillus sp. and 2 strains of Pseudomonas sp. Conclusions The results of the in vivo and the greenhouse experiments indicated that the selected isolates had a greatly significant effectiveness for suppressing blackleg and soft rot symptoms. The selected isolates could, therefore, be used as a biocontrol agent against blackleg and soft rot of potato.