Fruit rot caused by Fusarium species represents a significant threat to watermelon (Citrullus lanatus (Thunb.) Matsum. et Nakai) quality and economic value. In this study, a fungal pathogen designated strain X2 was isolated from rotten watermelon fruit and identified as Fusarium proliferatum (Matsush.) Nirenberg ex Gerlach & Nir through integrated morphological, microscopic, and multi-locus molecular phylogenetic analyses. Koch’s postulates were fully satisfied, confirming that F. proliferatum strain X2 induces water-soaking and softening symptoms on watermelon fruit at relatively high temperatures. However, low-temperature storage at 4 °C strongly suppressed fungal growth and symptom development. Strain X2 produced fumonisins (FB1, FB2, and FB3) in vitro, but all of these mycotoxins were below the limit of detection in infected fruit tissues. Dual-culture assay revealed that three antagonistic Bacillus strains (G3-2, KP3P9 and K13C) can significantly inhibit the growth of F. proliferatum strain X2. Consistently, fruit co-inoculation assays verified that these strains differentially alleviated external and internal rot symptoms caused by the fungus. Biochemically, F. proliferatum strain X2 infection markedly increased cell wall-degrading enzyme (CWDE) activities, whereas co-inoculation with biocontrol Bacillus bacteria suppressed the activities of polygalacturonase (PG), pectin lyase (PL) and cellulase (CL) in a strain-specific manner. This study identified F. proliferatum strain X2 as a causal agent of watermelon fruit rot and highlights the roles of low-temperature storage and Bacillus-mediated biocontrol in disease suppression.