Eucalyptus wood vinegar (WV) is a versatile natural product employed in several areas such as for agriculture, zootechnics, and veterinary applications. However, the product may contain contaminants such as polycyclic aromatic hydrocarbons, methanol, metals, and so on in its raw form. Thus, refining is required to remove them. Only after reaching a high-purity grade can WV be applied as a basis for pharmaceuticals, antimicrobials, and additives for animal husbandry. This work aimed to assess the effect of refining on the contaminant content of a WV produced on an industrial scale and traded as agricultural input in the Brazilian market. WV was refined by sequential vacuum distillations and had its contaminant contents assessed before and after refining. The following chemical classes were investigated: metals and other elements, insecticides, fungicides, and herbicides (carbamates, organochlorines, organophosphates, pyrethroids, and others), mycotoxins, polychlorinated dioxins and furans, polycyclic aromatic hydrocarbons, and volatile organic compounds, encompassing more than 100 chemical species. Also, the WV physical–chemical properties before and after refining were determined. Gas chromatography and mass spectrometry (GC/MS) were employed to attain the WV’s chemical profile. The main chemical components, such as phenolic compounds, furans, ketones, pyrans, etc., were annotated in the WV chemical profile. No traces of insecticides, fungicides, herbicides, heavy metals, mycotoxins, polychlorinated dioxins, or polychlorinated furans were found in raw WV or refined versions. However, several other undes/irable chemicals were determined in the raw product, such as PAHs and volatile organic compounds (acetaldehyde, acetone, ethylene glycol, and methanol). Nevertheless, all of them were effectively removed from the raw WV after the first or second sequential refining steps, resulting in a pure product. Therefore, only one refining step can be applied to raw WV to yield a high-quality product for pharmaceutical and animal husbandry applications.