Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor that binds to the CCAAT cis-element in the promoters of target genes and plays critical roles in plant growth, development, and stress responses. In the present study, we aimed to re-characterize the ClNF-Y family in watermelon, examine the assembly of ClNF-Y complexes, and explore their possible involvement in disease resistance. A total of 25 ClNF-Y genes (7 ClNF-YAs, 10 ClNF-YBs, and 8 ClNF-YCs) were identified in the watermelon genome. The ClNF-Y family was comprehensively characterized in terms of gene and protein structures, phylogenetic relationships, and evolution events. Different types of cis-elements responsible for plant growth and development, phytohormones, and/or stress responses were identified in the promoters of the ClNF-Y genes. ClNF-YAs and ClNF-YCs were mainly localized in the nucleus, while most of the ClNF-YBs were localized in the cytoplasm of cells. ClNF-YB5, -YB6, -YB7, -YB8, -YB9, and -YB10 interacted with ClNF-YC2, -YC3, -YC4, -YC5, -YC6, -YC7, and -YC8, while ClNF-YB1 and -YB3 interacted with ClNF-YC1. A total of 37 putative ClNF-Y complexes were identified, e.g., ClNF-YA1, -YA2, -YA3, and -YA7 assembled into 13, 8, 8, and 8 ClNF-Y complexes with different ClNF-YB/-YC heterodimers. Most of the ClNF-Y genes responded with distinct expression patterns to defense hormones such as salicylic acid, methyl jasmonate, abscisic acid, and ethylene precursor 1-aminocyclopropane-1-carboxylate, and to infection by the vascular infecting fungus Fusarium oxysporum f. sp. niveum. Overexpression of ClNF-YB1, -YB8, -YB9, ClNF-YC2, and -YC7 in transgenic Arabidopsis resulted in an earlier flowering phenotype. Overexpression of ClNF-YB8 in Arabidopsis led to enhanced resistance while overexpression of ClNF-YA2 and -YC2 resulted in decreased resistance against Botrytis cinerea. Similarly, overexpression of ClNF-YA3, -YB1, and -YC4 strengthened resistance while overexpression of ClNF-YA2 and -YB8 attenuated resistance against Pseudomonas syringae pv. tomato DC3000. The re-characterization of the ClNF-Y family provides a basis from which to investigate the biological functions of ClNF-Y genes in respect of growth, development, and stress response in watermelon, and the identification of the functions of some ClNF-Y genes in disease resistance enables further exploration of the molecular mechanism of ClNF-Ys in the regulation of watermelon immunity against diverse pathogens.