Abstract Background Single nucleotide polymorphisms (SNPs) play a significant role in differences in individual’s susceptibility to diseases, and it is imperative to differentiate potentially harmful SNPs from neutral ones. Defensins are small cationic antimicrobial peptides that serve as antimicrobial and immunomodulatory molecules, and SNPs in β-defensin 1 (DEFB1 gene) have been associated with several diseases. In this study, we have determined deleterious SNPs of the DEFB1 gene that can affect the susceptibility to diseases by using different computational methods. Non-synonymous SNPs (nsSNPs) of the DEFB1 gene that have the ability to affect protein structure and functions were determined by several in silico tools—SIFT, PolyPhen v2, PROVEAN, SNAP, PhD-SNP, and SNPs&GO. Then, nsSNPs identified to be potentially deleterious were further analyzed by I-Mutant and ConSurf. Post-translational modifications mediated by nsSNPs were predicted by ModPred, and gene-gene interaction was studied by GeneMANIA. Finally, nsSNPs were submitted to Project HOPE analysis. Results Ten nsSNPs of the DEFB1 gene were found to be potentially deleterious: rs1800968, rs55874920, rs56270143, rs140503947, rs145468425, rs146603349, rs199581284, rs201260899, rs371897938, rs376876621. I-Mutant server showed that nsSNPs rs140503947 and rs146603349 decreased stability of the protein, and ConSurf analysis revealed that SNPs were located in conserved regions. The physiochemical properties of the polymorphic amino acid residues and their effect on structure were determined by Project HOPE. Conclusion This study has determined high-risk deleterious nsSNPs of β-defensin 1 and could increase the knowledge of nsSNPs towards the impact of mutations on structure and functions mediated by β-defensin 1 protein.