Visibly distressed, Saudi-based star Neymar left the pitch on a stretcher after receiving treatment for several minutes.
The 31-year-old was making his 128th appearance for Brazil, and without him the five-time world champions had their worst performance under new coach Fernando Diniz, who will remain in the job at least until next year's Copa America.
Brazil's Neymar is carried off during a 2-0 World Cup qualifying loss to Uruguay in Montevideo.
Brazil FA sources said the forward had sustained a severe left knee sprain and that he would undergo tests to determine if there was ligament damage.
"In the short run we need to be realistic, we need to improve," Brazil captain Casemiro said.
"For Neymar to leave the match it is surely something serious. But I hope it isn't at the end. He has had those injuries whenever he starts picking up his pace again, it is hard."
The victory lifted Uruguay to second in the CONMEBOL standings on seven points, level with Brazil and Venezuela, who earlier shocked Chile 3-0.
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In Montevideo, Nunez opened the scoring in the 42nd minute with a close-range diving header and Brazil's night went from bad to worse two minutes later when Neymar went down after tussling for the ball with de la Cruz.
De la Cruz extended the hosts' lead in the 77th with a close range effort, with Nunez providing the assist to inflict on Brazil a first defeat in 37 matches of World Cup qualifying.
Elsewhere, Ecuador and Colombia played out a scoreless draw and Paraguay beat Bolivia 1-0 with a goal by Antonio Sanabria in the 69th minute.
Salomon Rondon scored his 46th goal for Venezuela in their 3-0 World Cup qualifying win over Chile.
In Maturin, hosts Venezuela opened the scoring shortly before the break through Yeferson Soteldo. Salomon Rondon added a goal in the 72nd minute and Darwin Machis put the final number on the scoreboard seven minutes later. Last week, Venezuela had a 1-1 draw against Brazil.
Leaders Argentina are in action away to Peru later on Tuesday night (Wednesday AEDT).
The next World Cup in the United States, Mexico and Canada will include 48 teams, meaning direct entry for the top six teams in South America. The seventh-placed team will contest an intercontinental playoff for a berth.
With AP.
