The increase in food production to meet the demand of the world’s growing population is largely conditioned by the efficiency of agricultural fertilizers. The development of relatively low-cost urease inhibitors and more environmentally friendly ones is a challenge for agriculture, especially because urea is the most used nitrogen fertilizer globally. The pyroligneous acid (PA), a by-product of wood charcoal production, stimulates plant growth, increases crop productivity, improves microbial metabolism, and induces plant tolerance to (a)biotic stresses. This work investigated the in vitro urease inhibitory potential of tree PAs of distinct plant biomass origin and production process. Coconut (Cocus nucifera) shell PA showed the highest urease inhibitory activity, followed by black wattle (Acacia mearnsii) PA and eucalyptus (Eucalyptus urograndis, a hybrid of Eucalyptus grandis and Eucalyptus urophylla). The same sequence was observed for the content of phenols and humic substances (humic acids + fulvic acids). This fact probably justifies the greater inhibitory activity, since both humic substances and some phenols normally present in PA, such as hydroquinone, have already been reported in the literature as urease inhibitors. The results are promising in terms of urease inhibition by PA samples, especially due to its low cost, biodegradability, non-toxicity, high solubility in water, and beneficial effects on plant development.