Born as the second son of Mori Yoshinari, Nagayoshi became head of the Mori clan upon the deaths of both his father and older brother in battle in 1570. He served Oda Nobunaga and participated in the attack and annihilation of the Takeda clan in 1582, capturing Iida Castle and Takatoo Castle in hard-fought battles. He was awarded a 20,000-koku fief of 4 districts of Kawanakajima in Shinano as well as Uozu Castle.

In June of that year, the situation in Shinano became unstable upon Nobunaga's death, and Nagayoshi fled back to his former stronghold, Kaneyama Castle in Mino. He died fighting against Tokugawa's forces in 1584 (it was said that he died instantly from a rifle bullet between the eyes). His younger brother Mori Tadamasa became the next clan head.