Since 2006, a bacterial spot disease has been widely observed on pumpkin and squash fruit in the North Central region (NCR) of the United States. Surveys were conducted during 2011 to 2013 to determine the occurrence of the disease on pumpkin and winter squash in the NCR. In each state, at least five commercial pumpkin and squash fields were visited each year within 3 weeks of harvest, and 60 fruit were inspected in each field. In nine states surveyed, 217 of 245 fields had fruit with bacterial spot. The average incidence of the symptomatic fruit in all pumpkin fields surveyed was 27.1, 30.3, and 25.3% in 2011, 2012, and 2013, respectively. The average incidence of fruit with bacterial spot in all squash fields surveyed was 20.2% in 2012 and 16.9% in 2013. The incidence of symptomatic fruit was more than 90% in some fields. Xanthomonas cucurbitae and non-X. cucurbitae bacteria were isolated from all of the symptomatic fruit collected. X. cucurbitae was identified based on colony characteristics on yeast extract-dextrose-CaCO₃ agar, a polymerase chain reaction test using primers RST2 and RST3, and pathogenicity on pumpkin leaves. All X. cucurbitae isolates tested were pathogenic on ‘Howden’ pumpkin. In total, 312 non-X. cucurbitae isolates were identified into 10 genera based on the restriction fragment length polymorphism analysis. None of the non-X. cucurbitae isolates was pathogenic on Howden. In vitro studies of interactions between non-X. cucurbitae and X. cucurbitae showed that Pantoea isolates had antagonistic activity against X. cucurbitae. Identified Pantoea agglomerans showed the highest antagonistic activity against X. cucurbitae.