Pathogenesis-related (PR) proteins are involved in plant responses to biotic and abiotic stress and are grouped into 17 families (PR1-PR17). The PR-5 family, also known as osmotins, comprises several proteins with different functions, including confirmed antifungal in vitro and in planta activity for most of them. A PR-5 gene was isolated by PCR from the black nightshade (Solanum nigrum L. var. americanum) genome, a solanaceous weed. The predicted mature protein is 207 amino acids in length and contains 16 cysteines involved in 8 disulfide bonds. This gene was named Solanum nigrum osmotin-like protein (SnOLP). In order to investigate and characterize the antifungal activity for the SnOLP protein, a His-tagged mature SnOLP form was overexpressed in Escherichia coli M15 strain carried out by a pQE30 vector construction. The solubilized His-tagged mature SnOLP protein was affinity-purified by immobilized-nickel affinity column chromatography. Renaturation mediated by reduced:oxidized gluthatione redox buffer generated biologically active conformations of the recombinant mature SnOLP, which exerted antifungal action towards the plant pathogenic Ascomycete anamorphs fungi Fusarium solani f. sp. glycines, Colletotrichum gossypii var. cephalosporioides, Colletotrichum gloeosporioides, Macrophomina phaseolina and against the plant pathogenic Oomycete Phytophthora nicotiana var. parasitica, under in vitro conditions. The in vitro mycelial growth inhibition of these economically important pathogens of soybean, cotton, stylosanthes and citrus crops, even at low doses of SnOLP (1 – 3 mg range), is an indicative that SnOLP represents a potent version of wild Solanum PR-5 protein with promising utility in biotechnological applications.