Plant defensins (PDFs) are a group of cationic antimicrobial peptides that are distinguished by their unique tertiary structure and play significant roles in physiological metabolism, growth, and stress tolerance. Defensins are key components of plant innate immunity; they can target a wide variety of microorganisms. This study aimed to identify and investigate the role of Triticum durum PDFs (TdPDFs) in response to environmental stresses. Prior to this, in silico analyses of TdPDF genes were conducted to assess their chromosomal locations, conserved motifs, exon–intron distribution, and cis-regulatory elements in the promoter regions. Additionally, bioinformatic analyses were performed to characterize the structure of TdPDF proteins, evaluate their phylogenetic relationships, predict their subcellular localization, and estimate their physicochemical properties. Docking studies were conducted to assess the interactions between TdPDF proteins and the fungal plasma membrane. A total of 28 TdPDF genes were identified in durum wheat based on their conserved domain PF00304 (gamma-thionin). These genes are distributed across all chromosomes of the durum wheat genome, except for chromosomes 4A and 7A. Analysis of the promoters of these genes revealed numerous elements associated with development, hormone responsiveness, and environmental stress. The majority of TdPDF proteins were predicted to be located extracellular. In addition, TdPDF proteins were classified into three clusters based on sequence similarity. Phylogenetic analysis suggested that TdPDF proteins share ancestral similarities with the PDF sequences of other monocotyledonous species. Molecular docking studies revealed that TdPDF proteins interact with fungal plasma membranes, suggesting that they play a critical role in the resistance of plants to pathogen infections. Expression analysis underlined the crucial role of nine TdPDF genes in the defense responses of durum wheat against both pathogenic and environmental stressors. Overall, our findings underscore the potential of TdPDF genes in host-plant resistance and highlight opportunities for their application in crop improvement toward stress tolerance.