During 1883-87 Niederle had studied at the Charles University in Prague. He was initially interested in classical archaeology, then studied anthropology, sociology and ethnology. Later, he studied in Munich under professor Johannes Rank (1889) and in Paris under professor Léonce Manouvriere at the École d’anthropologie. Niederle also travelled in several Slavic countries, studying archaeological findings and historical documents.
In 1898 Niederle was named professor at the Charles University. As archaeologist he had represented the "university school" (univerzitní škola), opposed to the "museum school" (muzejní škola) represented by archaeologist Josef Ladislav Píč.
During 1907-08 Niederle served as a dean of Faculty of Philosophy, during 1908-09 as a vice-dean and during 1927-28 as a rector of the faculty. In 1919 he helped to establish State Archaeological Institute (Státní archeologický ústav), today's Institute of Archaeology (Archeologický ústav) . He also published many articles about Slavic ethnography and archaeology and was editor of several specialised journals. Niederle had helped to set up Slavic Institute (Slovanský ústav) in Prague  whose director he was during 1928-31.
Among most known works are "Handbook of Czech Archaeology" (Rukověť české archeologie, 1910, with Karel Buchtela) and mainly the eleven volume series "Slavic Antiquities" (Slovanské starožitnosti) published between 1902-34. This series had exhaustively investigated origin and prehistory of the Slavs, continuing earlier work by historian Pavel Josef Šafařík.