In countries with high levels of urbanization, protected areas are often subject to human disturbance. In addition to dealing with fragmentation, land managers also have to confront the loss of characteristic ecosystems due to biotic homogenization, which is the increasing similarity of species assemblages among geographically separate regions. Using ants as a model system, we explored whether anthropogenic factors negatively affect biodiversity of protected areas of a regional network. We first analysed the effect of fragmentation and human activity on ant biodiversity within protected areas. Secondly, we tested whether homogenization could occur among protected areas. We sampled 79 plots in the most common habitats of 32 protected areas in southern Spain and calculated ant community richness and diversity indices, endemic richness, and Bray–Curtis similarity indices (between pairs of plots). We related these indices with patch fragmentation and human disturbance variables, taking into account environmental, spatial and landscape covariates. We used ANOSIM to test for differences between similarity indices, specifically among levels of anthropogenic disturbance. Species richness was positively correlated with the distance from the border of the protected areas and the number of endemic species was negatively correlated with the degree of fragmentation. Ant communities were similar within each protected area but differed across regions. Human disturbance was not correlated with community similarity among sampling points. Our approach suggests how the ability of European protected areas to sustain biodiversity is limited because they remain susceptible to anthropogenic impacts. Although ant communities maintained their biological distinctiveness, we reveal how fragmentation within protected areas is important for community richness and endemism maintenance.