The Winkler extraction is one of the two fundamental sampling techniques of the standardized “Ants of the Leaf Litter” protocol, which aims to allow qualitative and quantitative comparisons of ant (Hymenoptera: Formicidae) assemblages. To achieve this objective, it is essential that the standard 48—hour extraction provides a reliable picture of the assemblages under study. Here, we tested to what extent the efficiency of the ant extraction is affected by the initial moisture content of the leaf litter sample. In an Ecuadorian mountain rainforest, the leaf litter present under rainfall—excluded and rainfall—allowed plots was collected, its moisture content measured, and its ant fauna extracted with a mini—Winkler apparatus for a 48—hour and a 96—hour period. The efficiency of the Winkler method to extract ant individuals over a 48—hour period decreased with the moisture content of the leaf litter sample. However, doubling the extraction time did not improve the estimations of the ant species richness, composition, and relative abundance. Although the moisture content of the leaf litter slightly affected the ant sampling, our results indicated that a 48—hour Winkler extraction, as recommended by the “Ants of the Leaf Litter” protocol, is sufficient to allow reliable comparisons of ant assemblages.