Plant colonization on archaeological structures is limited by the substrate conditions of stone surfaces. Despite its relevance for ecological studies and the management plans of archaeological sites, the knowledge on monuments substrate (e.g., stones, microhabitats, microsites), which could be suitable sites for plant establishment, is an underestimated topic. Here, we aim to identify the substrate preference of plants in colonizing stone surfaces as a first step in evaluating the biodeterioration risk. We selected the Pasargadae World Heritage Site (WHS) for its variety of different conditions, and we assessed the substrate preference of 142 ruderal plants occurring on nine monuments, two types of stones, and eight microsites. Our data indicated that stone typology and microsite conditions affected species composition, diversity, and richness. The porosity of the different limestone and the progressive weathering phenomena resulted as relevant factors in plant colonization. Significantly, microsites as a junction of two stone blocks (M1a) and big fractures covered by soil (M3) hosted the most favorable conditions, which needed periodic monitoring during the maintenance activities of the site. Evaluating the substrate preference of ruderal plants can contribute to the identification of the high-risk areas for plant colonization, which need to be considered in the management plans of archaeological sites.