Pokkah boeng, once a minor disease is becoming major disease of sugarcane a total of 14 bacterial isolates were obtained from sugarcane phylloplane using HiCrome Bacillus Agar and characterized culturally for colony colour, shape and number of colonies per plate. Grams’ test differentiated the Gram-positive bacteria from Gram negative bacteria which was done using 3% KOH test. All these isolates were evaluated for their antagonism against Fusarium sacchari through dual culture technique. Out of 14 isolates, ScANKP-9 and ScANKP-13 isolates were found to be the most effective with maximum antagonistic potential by significantly restricting the growth of F. sacchari to 4.47 and 4.80 cm, in comparison to monocultured F. sacchari (9 cm) with 50.33 per cent and 46.67 per cent inhibition in radial growth. Molecular characterization of effective isolates using modified CTAB method following PCR amplification of DNA with FGPS-63 and FGPL-132-38 primers, BLAST and phylogenetic analysis confirmed the isolates, ScANKP-9 and ScANKP-13 as Bacillus amyloliquefaciens and Pseudomonas fluorescence, respectively.