Agricultural intensification in tropical landscapes poses a new threat to the ability of biological corridors to maintain functional connectivity for native species. We use a landscape genetics approach to evaluate impacts of expanding pineapple plantations on two widespread and abundant frugivorous bats in a biological corridor in Costa Rica. We hypothesize that the larger, more mobile Artibeus jamaicensis will be less impacted by pineapple than the smaller Carollia castanea. In 2012 and 2013, we sampled 735 bats in 26 remnant forest patches surrounded by different proportions of forest, pasture, crops and pineapple. We used 10 microsatellite loci for A. jamaicensis and 16 microsatellite loci for C. castanea to estimate genetic diversity and gene flow. Canonical correspondence analyses indicate that land cover type surrounding patches has no impact on genetic diversity of A. jamaicensis. However, for C. castanea, both percentage forest and pineapple surrounding patches explained a significant proportion of the variation in genetic diversity. Least-cost transect analyses (LCTA) and pairwise G″st suggest that for A. jamaicensis, pineapple is more permeable to gene flow than expected, while as expected, forest is the most permeable land cover for gene flow of C. castanea. For both species, LCTA indicate that development may play a role in inhibiting gene flow. The current study answers the call for landscape genetic research focused on tropical and agricultural landscapes, highlights the value of comparative landscape genetics in biological corridor design and management and is one of the few studies of biological corridors in any ecosystem to implement a genetic approach to test corridor efficacy.