Relationships among picoplankton, protozoa, phytoplankton, plant nutrients, lake type, drainage basin morphology and land cover were studied in 45 water bodies in South Island, New Zealand that ranged from large, deep, ultra-oligotrophic lakes to shallow, macrophyte-dominated ponds and swamps. The biomasses of most heterotrophic components of the pelagic microbial food webs were positively related to phytoplankton and features of the drainage basin that enhanced nutrient input, and imply strong resource-driven structuring of pelagic microbial food webs. Prokaryotic picophytoplankton biomass was negatively related to indices of eutrophication, and the picoautotroph contribution to total microbial food web biomass declined with increasing total phosphorus concentration from 16.5% in deep lakes to <0.02% in swamps and ponds. Biomass ratios of (picoplankton plus protozoa):phytoplankton ranged from 40:60 in swamps and ponds to >70:30 in deep lakes, and indicate the potential importance of microbial food webs in carbon transfer to higher trophic levels in deep, less productive lakes. Strong relationships exist between land use in the catchment and pelagic microbial food web structure and biomass across a wide range in size and trophic state of water bodies in heterogeneous landscapes.