Man-made karst ponds are the only surface water bodies in the Karst region (SW Slovenia, NE Italy) and support variable biotic communities, depending on ecological parameters and human activities. The aim of our study was to establish the relationship between environmental conditions (land-use of the catchment, vegetation of the banktop zone, morphological properties of the ponds, water quality parameters, the presence of fish) and aquatic macrophyte species composition and abundance. The survey of 28 ponds was performed in the peak vegetation period in 2009. The most abundant emergent macrophytes found were Typha latifolia, Alisma plantago-aquatica, Eleocharis palustris agg., and Alisma lanceolatum, while the most common hydrophytes were Lemna minor, Chara spp., Nymphaea alba, and Potamogeton natans. About 15% of all species found in ponds are included in the “Red” list of endangered plant species of Slovenia. The presence of fish, water transparency, pH, land-use of the pond catchment and quality of shore substrate were detected as the most influential factors determining aquatic macrophyte communities.