Anthropogenic fires pose a serious threat to many terrestrial ecosystems because they can cause loss of biodiversity and carbon stocks in the biosphere. Specifically, wildfires impacting natural conservation areas such as European Natura 2000 sites (N2K) are of particular concern. The main study objective was to evaluate the long-term effects of wildfires on the organic layer and some physical, chemical and biological properties of the underlying soil mineral layer, linked to soil quality. Here, we studied two coastal Mediterranean Aleppo pine stands within an N2K site differing for the fires’ years of occurrence, the time between fires (TBF) and the time since last fire (TSLF) throughout 24 years. Furthermore, in each stand, differences in fire frequency (FF) were considered by selecting three sites—double-fire, single-fire and control (unburnt). Our results show the absence of the O-layer in double-fire sites, indicating a loss of this organic carbon (if compared to control) pool of 204 g m−2 in R2F and 139 g m−2 in M2F. Despite this loss being offset by the Corg increase in soil mineral layer, the disappearance of O-layer may compromise the ecosystem services provided by soil. In each stand, long-term fire effects were evident at both single-fire and double-fire sites for some chemical as well as biological soil properties and depended on TSLF. Increased rates of nitrogen mineralization and nitrification were found at all burned sites, persisting up to 24 years post-fire. Soil quality indicators data highlighted the recovery handicap of the microbial community within the considered period. Since our outcomes showed wildfires enduring consequences, mainly relating to TSLF and FF, on different organic and mineral soil properties, we advocate employing prompt strategies to mitigate recurring fires.