The Vishwa Hindu Parishad, or World Hindu Council, a nationalist group linked to Prime Minister Narendra Modi's ruling party, acted after winning a court order to stop the killing of bulls and bullocks.
"Members of the VHP came to Deonar abattoir late at night and asked us to stop the slaughter, showing the copy of the High Court order," said Mohammad Ali Qureshi, president of the Bombay Suburban Beef Dealers Association.
Deonar, on the outskirts of Mumbai, is India's largest abattoir. The beef trade is mainly controlled by minority Muslims, raising concern that the ban is driven by Modi loyalists pursuing a Hindu agenda.
President Pranab Mukherjee last week gave his assent to the Maharashtra Animal Preservation Bill, which had been pending for 20 years, extending a ban on the killing of cows, considered sacred by Hindus, to bulls and bullocks.
The law calls for up to five-years jail for anyone found in possession of beef, according to media reports.
But the state government - led by a Modi's Bharatiya Janata Party - said it could take a week to implement the law, prompting the VHP to petition the Bombay High Court on Tuesday for an immediate ban.
"Why wait for another 5 to 6 days, just for paper formalities and the final draft?" asked Vyankatesh Abdeo, all-India secretary of the VHP. "If we would have waited thousands of cattle would have been slaughtered."