Leaving Harvard after two years in 1938, Seeger hitchhiked and rode freight trains around the country, gathering country ballads, work songs, and hymns and developing a remarkable virtuosity on the five-string banjo. In 1940 he organized the Almanac Singers, a quartet that also featured the folk singer and composer Woody Guthrie, and appeared at union halls, farm meetings, and wherever his populist political sentiments were welcome. The group disbanded soon after World War II.

In 1948 he formed another group, the Weavers (Weavers, the)—with Lee Hays, Ronnie Gilbert, and Fred Hellerman—that achieved considerable success on college campuses, in concert, and on several records. Shortly after the group had achieved national fame, however, a great deal of controversy was stirred up concerning Seeger's previous activities in left-wing and labour politics, and the Weavers suddenly found themselves blacklisted by much of the entertainment industry. Finding it increasingly difficult to make concert bookings or to sell records, the group broke up in 1952 but reunited three years later when a Christmas concert at Carnegie Hall sparked new interest in their music and message. Seeger left the group in 1958, and it disbanded in 1963. (The Weavers gave two reunion concerts in 1980, and a motion picture documentary about the group, Wasn't That A Time!, was released in 1982.) After the 1950s Seeger usually worked alone or with his family. As a solo performer he was still a victim of blacklisting, especially after his 1961 conviction for contempt of Congress stemming from his refusal in 1955 to answer questions posed to him by the House Committee on Un-American Activities concerning his political activities. Although Seeger's conviction was overturned the following year in an appeal, for several years afterwards the major networks refused to allow him to make television appearances. In later years the controversy surrounding the performer gradually subsided.

A beloved fixture at folk festivals, Seeger was given major credit for fostering the growth of the hootenanny (a gathering of performers playing and singing for each other, often with audience participation) as a characteristically informal and personal style of entertainment. Among the many songs that he wrote himself or in collaboration with others were “Where Have All the Flowers Gone,” “If I Had a Hammer,” “Kisses Sweeter Than Wine,” and “Turn, Turn, Turn.” His The Incompleat Folksinger (1972) is a collection of his writings on the history of folk songs, civil rights, and performers in his lifetime.

In the 1970s and '80s he was active in a program to remove pollution from the Hudson River, building the Hudson River sloop Clearwater, promoting festivals for its maintenance, and engaging in environmental demonstrations, particularly antinuclear ones. His father was the influential musicologist Charles Seeger, and his uncle the poet Alan Seeger.