Oleksiy Vadatursky, 74, and his wife Raisa died when a missile hit their home overnight, local officials said.
Mr Vadatursky owned Nibulon, a company involved in grain exports. He had also received the “Hero of Ukraine” award.
Mykolaiv mayor Oleksandr Senkevych said it was probably the heaviest Russian bombardment of the city so far.
There was damage to a hotel, a sports complex, two schools and a service station, as well as homes.
Mykolaiv is on the main route to Odesa, Ukraine’s biggest port on the Black Sea, and has been hit repeatedly since Russia launched its invasion on 24 February.
The region’s leader Vitaliy Kim said Mr Vadatursky’s “contribution to the development of the agricultural and shipbuilding industry, the development of the region is invaluable”.
Meanwhile, an adviser to Ukrainian President Volodymyr Zelensky’s office, said he believed Russia had deliberately targeted the businessman.
Mykhailo Podolyak said one of the missiles hit the businessman’s bedroom, adding that this “leaves no doubt” it was being guided.
Nibulon has built many storage facilities and other infrastructure for exporting grain.
Ukraine and Russia are major exporters of wheat and other grains, and the disruption of exports caused by the war has sent food prices soaring worldwide.
The two countries signed a UN-brokered agreement in Turkey last week, aimed at easing the food crisis. The deal was almost derailed by a Russian attack on the Odesa port the following day.
The resumption of Ukrainian exports has been further delayed by security checks. But on Sunday Turkey said the first ship carrying grain was expected to leave Odesa on Monday morning.
Ukraine accuses Russian forces of stealing grain from farms on occupied land and exporting it via Crimea – Ukraine’s southern peninsula annexed by Russia in 2014. Russia denies Ukrainian claims.
Blow to Russian Navy Day
Meanwhile, Russia cancelled Navy Day celebrations in occupied Crimea.
The reason given by Sevastopol Governor Mikhail Razvozhayev was an alleged Ukrainian drone strike on the Black Sea Fleet headquarters. The fleet has long been based in Sevastopol.
A senator in Russian-occupied Crimea later said a drone had been launched from within Sevastopol, and the perpetrators would be found by the evening.
Senior Ukrainian official Serhiy Bratchuk dismissed Russia’s claims as a “provocation”.
“The liberation of our Crimea will take place differently and much more efficiently,” he said.
A photo posted by Governor Razvozhayev showed him in a courtyard littered with leaves, but with no obvious structural damage. He said the Navy Day celebrations were cancelled for security reasons.
Navy Day is an annual Russian holiday, and celebrations are taking place across Russia. President Vladimir Putin is overseeing events in St Petersburg, his home city.
He signed a new naval doctrine naming the US as Moscow’s main rival, and also setting out the Kremlin’s global maritime ambitions for key areas such as the Arctic and in the Black Sea.
In a blow to the Black Sea Fleet, in April, its flagship Moskva sank after what Ukraine described as a strike with two Neptune missiles.
Russia admitted there was a big fire on board caused by exploding ammunition, without attributing it to a Ukrainian attack, and said the missile cruiser sank in a storm while being towed.
It remains unclear how many Russian sailors were killed or injured in the ship’s demise.
Meanwhile in the north, Ukraine’s second city Kharkiv was hit by Russian missiles again, mayor Ihor Terekhov said. Three Russian S-300 missiles struck a school there, destroying the main building, he said.
The BBC was unable to verify the latest reports.
In a late-night address on Saturday, Ukraine’s President Volodymyr Zelensky told all civilians still living in parts of eastern Donetsk region under Ukrainian control to evacuate.
The region has seen heavy clashes amid a slow advance by Russian forces, who already control large parts of it.
“The more people leave Donetsk region now, the fewer people the Russian army will have time to kill,” Mr Zelensky said. “We will use all available opportunities to save as many lives as possible.”
