Trilobite biofacies are often indicative of particular paleoecological settings and can reveal spatial paleogeographic changes. Multivariate analyses (CA, PCoA and NMDS) were performed using the Raup-Crick coefficient on a dataset of 62 trilobite genera and subgenera from 15 localities across South China during the Early–Middle Ordovician (Floian–Dapingian). Seven trilobite biofacies are distinguished from platform to slope environments, showing prominent ecological differentiation along bathymetric gradients and across different substrate conditions. Benthic and endemic trilobites generally occurred on the shallow-water platform, whereas the mesopelagic and atheloptic forms were associated with graptolite-dominated slope facies. The Olenid Biofacies and the Cyclopygid Biofacies, identified from deep-water environments, support the existence of the Jiangbei Slope (Northern Slope) and the Jiangnan Slope (Southern Slope) in South China at least during the Early–Middle Ordovician.