Mango anthracnose, caused by Colletotrichum gloeosporioides, is a significant disease affecting mango production. This study aimed to develop and evaluate a bio-formulation of Serratia nematodiphila GCSR38 for controlling mango anthracnose. The antagonistic activity of S. nematodiphila GCSR38 was tested using the dual culture method, achieving a 40.21% inhibition rate against C. gloeosporioides. Three powder formulations were developed using a low-heat drying process, differing in their composition of corn starch, talcum, calcium carbonate, and carboxymethyl cellulose (CMC). The formulations exhibited varying physical characteristics, water solubility, and moisture stability, with pH values ranging from 7.90 to 8.00. The survival of S. nematodiphila GCSR38 in the powder formulations remained substantial over 120 days, with final bacterial counts between 2.50 × 107 and 5.10 × 107 CFU/g. Efficacy testing on mango leaves showed that Bio-Formulation 3 provided the highest inhibition of C. gloeosporioides, achieving 51.00% inhibition in preventive application and 48.30% in curative application. These results were comparable to the chemical fungicide Carbendazim (56.11% and 49.31%, respectively), with no significant differences observed. Preventive application proved more effective than curative treatment. Overall, S. nematodiphila GCSR38 shows promise as a biocontrol agent for mango anthracnose, offering an eco-friendly alternative to chemical fungicides.