SH3 (Src homology-3) domain-containing proteins are conserved molecular scaffolds that mediate protein-protein interaction and regulate important cellular processes in eukaryotes. However, their role in phytopathogenic fungi remain poorly understood. In this study, we systematically identified and functionally characterized SH3 domain-containing proteins in the plant fungal pathogen Fusarium graminearum. We identified 29 SH3 domain-containing proteins in F. graminearum, of which only 9 were previously characterized. We found that the remaining 20 proteins, including FgSla1, FgAip5, FgRax2, FgMcy1, FgVta, FgPin3, FgYsc84, FgSh3A, FgSh3B, FgSh3C, FgBoi1, FgRvs167, FgBzz1, FgClf, FgCyk3 and FgHof1, are required for vegetative growth, plant infection and deoxynivalenol (DON) production. Notably, the absence of FgRAX2 and FgMCY1 completely abolished DON synthesis. FgHof1 and FgRax2 serve as positive and negative regulators of conidiation, respectively, and are indispensable for sexual development. Furthermore, FgHof1 and FgCyk3 are crucial for cytokinesis and nuclear distribution, as shown by irregular septation and nuclear fragmentation in the mutant strains. Subcellular localization revealed distinct distributions of these proteins, including the cytoplasm, septa/septal pore, plasma membrane, sub-apical collar and hyphal tip, consistent with the multifaceted functions of the proteins. Remarkably, FgHof1 localizes to septal pore and its deletion causes conidial breakage along the septa. FgAip5 localizes to the hyphal tip and its absence leads to retarded growth and irregular colony edges. Interestingly, several SH3 proteins contain intrinsically disordered regions (IDRs) and form protein condensates in the cytosol. These proteins exhibited features of phase separation like condensate fusion and reemergence after photobleaching, suggesting a possible role in dynamic protein assembly. Deletion of the IDRs largely altered these features in the proteins. In summary, this study highlights the varied functions of SH3 domain-containing proteins in growth, asexual/sexual development, DON biosynthesis and pathogenicity of F. graminearum, offering new insights into the functional diversity of SH3 proteins in fungal pathogenesis and potential targets for the control of Fusarium head blight (FHB).