In Serbia, almost all charophyte species belong to one of the IUCN categories. This study aimed to gather more knowledge about their floristic richness, distribution and ecology. During the survey, 262 localities were investigated in the Vojvodina province, northern Serbia. Seventeen charophyte species were found, out of which 16 were included in the analyses. The environmental matrix included 38 localities, each characterized by habitat type and 35 environmental parameters. The canonical correspondence analysis (CCA) showed six parameters as being statistically significant: substrate, altitude, depth, water temperature, temperature annual range and precipitation of the driest month. Together, these explain the 32.34% variability in the species composition and abundance data. Altitude and substrate contributed the most to this. Two main types of habitats were outlined by the analysis. Small and shallow temporary habitats in the river floodplains, at lower altitudes, with muddy and clayish substrate, belong to one type. These areas are inhabited by either very tolerant species, such as Chara vulgaris and C. globularis, or the so-called “spring” species of the genera Tolypella and Nitella. Habitats which are located at higher altitudes, when deeper and more permanent with a sandy substrate, such as sandpits and river habitats, belong to the second one. These areas are inhabited by species like Chara papillosa, C. hispida, and Nitellopsis obtusa.