Marquez, who had fought his way back through the field from 19th place after a ride-through penalty, appeared to mount a wildly misjudged attempt to pass his rival.
The Catalan's Repsol Honda collided with Rossi's Movistar Yamaha, shoving the Italian to the edge of the track and onto the wet grass run-off area.
To audible gasps from the stands, Rossi was sent sprawling. He remounted only to limp home in 19th place and out of the points. Marquez crossed the line in fifth, but a penalty for 'reckless riding' pushed him back to 18th, also pointless.
Rossi was damning in his assessment of both the incident and Marquez's race weekend overall, pointing to other incidents involving the Honda rider.
"One incident can happen to anybody, you can make a mistake in braking, you can touch the other guy. It can happen, it's racing," he told reporters.
"But from Friday morning on, Marquez did this to [Maverick] Viñales, to [Andrea] Dovizioso, to me and today he went straight through four riders."
His penalty following the clash with Rossi was the third for Marquez on a frenetic afternoon.
After stalling his bike on the grid there appeared to be confusion over whether he should start from the pit lane.
But Marquez mistakenly remained on the grid, storming to a commanding lead after robustly passing even his teammate Dani Pedrosa, who was also then sent dramatically tumbling by an overly-aggressive pass from Tech 3 Yamaha's Johann Zarco.
A ride-through penalty for Marquez's starting grid infraction was the beginning of his problems as he fought to regain places.
A first misjudged pass attempt saw him barge into Aprilia's Aleix Espargaro, for which race direction ordered him to drop a place, all before the calamitous incident with Rossi.
Marquez tried to explain himself after the race. "[After the ride-through penalty] I obviously tried to push to recover and, unfortunately, I made some mistakes, the biggest one I believe being with Aleix [Espargaro]," he told reporters.
"I approached faster than him and I didn't realize it. I tried my best to avoid contact but unfortunately I wasn't able."
The world champion blamed the track for his clash with Rossi. "With Valentino it was different, as it was a consequence of the track conditions," he said.
"I touched a wet patch, locked the front, and released the brakes. I tried to turn, again making my best effort to avoid contact."
Even without the drama of the Rossi-Marquez clash, the race was memorable enough. Cal Crutchlow became the first British rider to lead the championship since the legendary Barry Sheene in 1979, with a superbly judged win in capricious conditions.
The LCR Honda rider patiently tracked an unlikely leading group containing Zarco, Suzuki's Alex Rins and Pramac Ducati's Jack Miller, before launching a perfectly timed last lap bid for victory.
Crutchlow looks to have found his perfect ride this season. "She's a rocket when she needs to get going," he said of his bike.
"Now we come away from here leading the championship and we have to be positive going on to [the next race in] Austin."