The arrests were made late on Friday after police sealed the Kolkata office of IVRCL, the contractor behind the ill-fated construction project in West Bengal state.
Five other staff were detained for questioning over the tragedy that unfolded after a 100-metre section of the flyover crashed down on Thursday, crushing pedestrians and vehicles on the street below.
"Three mid-level officials of the Hyderabad-based construction firm were arrested last night," Kolkata police joint commissioner Debasish Boral told AFP.
"We have also detained five others of the firm, sealed its office in Kolkata and sent a notice to its managing director to join the investigation into the incident," he said.
The three arrested employees are expected to appear in court Saturday, where a public prosecutor will outline the initial charges against them.
Although officials have ruled out the chance of finding any more survivors under the rubble, a rescue operation continued Saturday at the site where blocks of concrete and twisted girders lay strewn.
The death toll rose to 26 after rescuers found the body of a truck driver's assistant lying crushed under the rubble, Boral said.
The West Bengal government has ordered an investigation into the state agency responsible for infrastructure and construction, over how the project came to be approved.
Police have registered a case of culpable homicide against the firm while Derek O'Brien, a state lawmaker, has said the company had been blacklisted in other states and had a "bad reputation".
Construction of the two-kilometre-long flyover began in 2009 and was supposed to be completed within 18 months, but suffered a series of hold-ups.
IVRCL has denied responsibility for the disaster in the capital of West Bengal state. A company representative infuriated victims on Thursday when he described the disaster as an "act of God".
Authorities initially struggled to get cranes and other large machinery through the narrow streets of Burrabazar, one of the oldest and most congested parts of the city where the flyover collapsed.
On Saturday, Rahul Gandhi, the vice-president of the opposition Congress party, visited the accident site and met with injured victims recuperating at a city hospital.
The disaster is the latest in a string of deadly construction accidents in India, where enforcement of safety rules is weak and substandard materials are often used.
The disaster comes at a sensitive time for West Bengal's Chief Minister Mamata Banerjee, whose centre-left Trinamool Congress party is seeking re-election.
Banerjee has blamed the previous state government under which the flyover project was started, but has herself faced criticism over the beleaguered construction project.
Voting in the West Bengal elections begins on Monday and will be held in five phases lasting a month.
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