The antagonistic potentials of endophytic bacteria isolated from the roots of six cotton cultivars at different developmental stages were determined in vitro toward three pathogens: Verticillium dahliae Kleb V107 and V396 and Fusarium oxysporum f.sp. vasinfectum (F108). The populations of antagonistic endophytic bacteria (AEB) toward V107, V396, and F108 at the flowering and maturation stages were significantly higher than those at the seedling stage were. More AEB were found to be antagonistic toward pathogens V396 and F108 than V107. Results from the multivariate analysis of variance showed that the populations of AEB were significantly different for the main factors of cultivars, stages, and their interactions. Based on 16S rDNA sequence analysis, the 39 AEB isolates that antagonized V107, V396, and F108 (BAEB) consisted of seven genera, in which the genus of Enterobacter (17 out of 39) and Pantoea (14 out of 39) were predominant among the BAEB isolates. Characterized by BOX-PCR fingerprints, these 39 BAEB isolates represented 35 different cluster types. To explore the antagonistic mechanisms, the agar diffusion method was used to detect cell-wall-degrading enzyme activity and siderophore secretion. Nearly half of these BAEB isolates showed protease and chitinase activity, while all 39 BAEB isolates excreted siderophores. However, pectinase, cellulase, and xylanase activity were hardly detected. A germination experiment revealed that nine of the 39 BAEB isolates significantly improved the vigor index of the cotton seedlings.