Ponds are biodiversity hotspots that harbor diverse macro-organisms and microbial communities, despite being isolated, highly variable environments with island-like features. These factors may give rise to unique biogeographic patterns and drivers of microbial community diversity. Here, we evaluated environmental and regional drivers of pond fungal and bacterial biodiversity to determine whether these communities follow commonly observed biogeographical trends. We collected water and muck from 40 ponds across 8 states in the Eastern USA and sequenced sections of the 16S rRNA gene and ITS1 region to survey the bacterial and fungal communities and test (1) whether these communities follow latitudinal diversity gradients and distance decay relationships, and (2) if variation in community composition or richness was related to specific environmental or land-use factors. We found that pond microbial communities exhibit distinct distribution patterns and relationships with environmental drivers depending on the microhabitat and microbial taxa. For example, muck fungal communities followed latitudinal diversity gradients, but bacterial communities did not. Abiotic conditions within ponds explained more variation than surrounding land use or the distance between sites, with temperature universally linked to microbial biodiversity. All sample types displayed weak distance-decay relationships, likely because of low spatial autocorrelation in environmental conditions and the lack of connectivity across ponds.