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The worldwide increased difficulty to counteract gastrointestinal nematode (GIN) infection in sheep, due to progressing anthelmintic resistance, has led to the evaluation of other alternative helminth control options, mainly from plants. The anthelmintic efficacy of an aqueous Punica granatum macerate was evaluated in sheep naturally infected by GIN in southern Italy. The macerate was chemically characterized by chromatographic analysis coupled with high-resolution mass spectrometry (LC/HRMS) and an aliquot was concentrated to obtain a dry extract. A part was characterized, the remaining washed with methanol to obtain an insoluble residue and methanol phase. In the methanol fraction, the quantitatively predominant gallic acid was purified to obtain the pure molecule. The three fractions thus obtained were used for in vitro studies (i.e., egg hatch test) to verify anthelmintic efficacy. For this purpose, fecal samples were collected from sheep naturally infected by GINs. Fractions were diluted in H2O/DMSO 0.5% at 1.00, 0.5, 0.25, 0.125, 0.05, and 0.005 mg/mL concentrations. Thiabendazole (0.25 and 0.5 mg/mL) and deionized water were used as positive and negative controls, respectively. Egg hatch test results indicated that all fractions caused a significant (p &lt, 0.05) egg hatch inhibition within 48 h of exposure highlighting a high (&gt, 82%) efficacy in vitro at all tested doses. Maximal egg hatching inhibition effect was exhibited by the methanol fraction (99.3% and 89.3% at 1 and 0.005 mg/mL concentrations), followed by the insoluble residue and gallic acid (94.7% and 85.3% and 94.0% and 82.7% at 1 and 0.005 mg/mL, respectively). The current study validated the anthelmintic potential of traditional P. granatum macerate against GIN infection in sheep, thus highlighting the role of gallic acid as principal component and justifying a need to undertake further in vivo studies on these ethno-veterinary remedies.