Yamauchi Masatsuna served as a bushō during the late-Muromachi period.  He was a retainer of the Rokkaku clan based in the southern portion of Ōmi Province.

The Yamauchi were a branch of the Rokkaku, members of the Sasaki clan descended from the Ōmi-Genji.  On 10/2 of Kōshō 2 (1456), Rokkaku Hisayori who suffered from the intervention of Kyōgoku Mochikiyo died in anger.  Thereafter, Masatsuna served as the guardian of Hisayori’s lineal heir, Kamejumaru (later known as Rokkaku Yukitaka and then Rokkaku Takayori).  In 1458, Kamejumaru was ousted by the Muromachi bakufu and his older cousin, Rokkaku Masataka, was appointed as the military governor of Ōmi and became the head of the Ōmi clan.  In 1460, after Masataka murdered the lineal heir of Iba Mitsutaka, he was removed from the line of succession and headship of the clan reverted to Kamejumaru.  Masatsuna loyally supported Kamejumaru.

From 1467, during the Ōnin-Bunmei War, Masatsuna and the Rokkaku clan allied with the Western Army led by Yamana Sōzen and fought against the Kyōgoku clan associated with the Eastern Army.  In the eleventh month of 1468, Masatsuna returned to Ōmi and served as a proxy for Yukitaka to strengthen defenses at Kannonji Castle in a bid to withstand an attack by the army of Kyōgoku Mochikiyo and Rokkaku Masataka.  Facing defeat, he set fire to the castle and fled.

After the Ōnin-Bunmei War, as Yukitaka aimed to strengthen his authority in pursuit of becoming a sengoku daimyō, he sought to control the powers of the noble families along with the temples and shrines.  After seizing the lands of nobles, temples and shrines, in addition to lands of the hōkōshū, or the military organ of the bakufu, he reallocated the territory to kokujin, or provincial landowners, under his command.  This was opposed by, among others, Ashikaga Yoshihisa (the ninth shōgun of the Muromachi bakufu), who, in 1487, led a campaign to Ōmi to subdue him, causing Yukitaka to flee to the mountains of Kōka.  This was the first phase of a campaign by the bakufu army against the Rokkaku clan known as the Chōkyō-Entoku Expedition.

In 1489, Yoshihisa died so the campaign was suspended.  Ashikaga Yoshiki (later known as Ashikaga Yoshitane; the tenth shōgun and successor to Yoshihisa) pardoned Yukitaka, so Yukitaka was restored to his position as the military governor of Ōmi.  Yukitaka, however, did not return the territory earlier seized and allocated to the kokujin, or provincial landowners, so, beginning in the eighth month of 1491, he was subject to another campaign led by Ashikaga Yoshiki, the tenth shōgun of the Muromachi bakufu, to eliminate him in the second phase of the Chōkyō-Entoku Expedition.  In the twelfth month, he abandoned his base, traversed the mountains of Kōka, and fled in defeat to Ise Province.  Yoshiki planned to back as the new military governor the adopted son of Rokkaku Masataka named Torachiyo or Hachirō who originated from the Takashima clan.

In the eleventh month of 1492, Yukitaka dispatched Masatsuna as a messenger of peace to Ashikaga Yoshiki at Onjō Castle in Ōtsu whereupon he was murdered by the army led by Shiba Yoshihiro and Akamatsu Masanori.  (Under another interpretation, he was summoned to the castle by Yoshiki.)

In the fourth month of 1493, Yoshiki was deposed in a coup d’ètat known as the Meiō Political Incident.  In the wake of this incident, in the tenth month Masatsuna’s son, Yamauchi Naritsuna, became the head of the Rokkaku clan and was appointed by Yoshiki’s successor, Ashikaga Yoshizumi, to serve as the military governor of Ōmi.