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.
Ukraine's President Volodymyr Zelensky described Mr Vadatursky's death as a great loss.
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 President 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.