Bacterial spot disease caused by Xanthomonas cucurbitae is among the most important foliar and fruit diseases of cucurbits worldwide. Given the absence of resistant cultivars, along with the limited effects of chemical treatments, we determined whether potential biocontrol agents could be effectively used to manage the disease. More than 2000 bacterial isolates associated with bacterial spot symptomatic fruits and leaves were collected and screened in vitro for their antagonistic activity against X. cucurbitae ATCC 23378. Of these, 241 isolates inhibited the growth of X. cucurbitae in a disc diffusion assay. These antagonistic isolates were preliminarily identified using 16s rRNA primers, and the species identity of promising strains was later determined using six housekeeping genes in a MLST analysis. Based on the in vitro assays, we found that the most effective isolates were Bacillus amyloliquefaciens, Pantoea agglomerans, and Pseudomonas putida, with all of these species having previously described biocontrol functions against other disease-causing bacteria in plants. In a greenhouse experiment, some of the isolates reduced bacterial spot disease symptoms and showed growth promoting effects on ‘Howden’ pumpkin (Cucurbita pepo). Our results suggest that some of these biocontrol agents may provide effective disease control, with the added advantage of plant growth promotion, when used under field conditions.