His formal education in music began with Josef Stastny at the Litomysl Gymnasium (1888–1896), alongside instruction in Czech history. In 1896 he moved to Prague to study at Charles University, where he attended lectures in positivist history with Jaroslav Goll and music aesthetics with Otakar Hostinský, finally receiving his doctorate in 1900.
Hostinský, a great proponent of Smetana's music, suggested that Nejedlý study composition and music theory with his like-minded colleague, Zdeněk Fibich, whose personality and tastes had a profound effect on his young student.
Although his first publications were devoted to Czech history, after Fibich's death in 1900 Nejedlý devoted himself to musicology, authoring a monograph entitled Zdenko Fibich, Founder of the Scenic Melodrama in 1901 as a first attempt at gaining greater recognition for his mentor. That these efforts were directed against the musical establishment of Prague (who he felt had victimized Smetana, Fibich, and Hostinský) was made clear by his first foray into music criticism that same year, in an attack on Antonín Dvořák's opera Rusalka shortly after its premiere.
His 1903 History of Czech Music drew distinct battle lines between the Conservatory students of Dvořák and the supposed inheritors of Smetana, including the composers Josef Bohuslav Foerster, Otakar Ostrčil, and Otakar Zich, all personal friends of Nejedlý's on the outs with the Prague establishment. Over the next decade he produced an extraordinary amount of writing on music, including monographs on pre-Hussite song (1904, 1907, and 1913), Smetana's operas 1908, Czech Modern Opera Since Smetana (1911, notoriously excluding Dvořák), Hostinský (1907 and 1910), and Gustav Mahler (1913).
In 1908 Zdenek Nejedly began to lecture in musicology at Charles University, forming a circle of devoted young colleagues that included Zich and Vladimír Helfert. When Nejedly's music reviews for Prague's daily newspapers grew distasteful in their anti-Conservatory bias, he and his followers were precipitously banned from publication, forcing the group to found their own journal, Smetana, which ran for sixteen years, 1910-1927. From this vantage point Nejedly launched the so-called "Dvorak Affair" (1911–1914), in which he sought to attack the legacy of the great composer; any contemporary artists who sided against him (especially the 31 musicians who signed a public petition in 1912) became the focus of fierce personal attacks. Beginning with Vitezslav Novak in 1913, Nejedl sought to end the careers of composers who did not conform to his pro-Smetana views of modern tradition and social responsibility: other notable targets included Josef Suk. Meanwhile, these tactics came back to haunt Nejedly's own proteges, especially Ostrčil as director of Prague's National Theatre and Zich as a modernist opera composer.
After the legalization of the Czechoslovak Communist Party in 1921, Nejedly became one of its earliest and most outspoken members. With the exception of his Smetana journal, he turned away from mainstream journal publications, focusing on the Communist daily Rude pravo and his own political journal, Var (Boiling, 1921–1930). In these he chastised the interwar Czechoslovak Republic, its president Tomas Garrigue Masaryk, and various other leaders; the last issue of Var was taken up with a detailed defense of Alban Berg's opera Wozzeck, which Ostrcil had produced in 1926.
During the Nazi occupation of the Czech Lands, the Nejedlý family fled to the Soviet Union, where he supposedly helped Czech resistance activities from afar. After the end of the war Zdeněk Nejedlý returned to Prague to participate in the postwar government. Initially he was made Minister of Education, Arts, and Sciences, but this was exchanged for Social Security by 1946. After the February Revolution of 1948 he returned to Culture and Education, a post he kept until 1953. These crucial years saw the implementation of a statewide curriculum at all levels of education.
Zdeněk Nejedlý died on March 9, 1962, and was buried in the Vyšehrad cemetery at Prague's Vyšehrad castle, reserved for Czech heroes and significant representatives of Czech culture. His grave is near those of Smetana, Ostrčil, and his son, Vít.