Puroindolines (PINs) are the main components of the wheat grain hardness locus (Ha) and have in vitro antimicrobial activity against bacteria and fungi. Here, we examined the effect of variation in PINA and/or PINB content upon Penicillium sp. seed fungal growth inhibition. The Penicillium sp. assays were germination assays performed after incubating seeds in Penicillium sp. contaminated soil. The first set of wheat genotypes consisted of two sets of transgenic isolines created in the varieties ‘Bobwhite’ and ‘Hi-Line’ having over-expression of PINA and/or PINB. The second set of genotypes consisted of near-isogenic lines (NILs) varying for mutations in PINA or PINB created in the varieties ‘Explorer’ and ‘Hank’. After incubation in Penicillium sp.-infected soil, transgenic wheat seeds over-expressing PINA in both ‘Hi-Line’ and ‘Bobwhite’ and both PINs in ‘Hi-Line’ exhibited significantly reduced fungal infection and increased germination. No significant differences in Penicillium sp. infection or germination rates were observed in seeds of the NILs. The results indicate that puroindolines native role in seeds is to increase seed viability and that when over-expressed as transgenes, the puroindolines are effective antifungal proteins.