Suspected militants set off a series of car bombs on Saturday that killed 14 people and wounded hundreds in the deadliest attacks to hit the region in recent years.
"There are about 300 of leader rank, 3,000 operators and about 10,000 supporters," the general said, adding that their numbers had fallen due to arrests.
Prayut called for people to remain calm following the weekend attacks in which a tourist hotel was targeted, raising concerns as Thailand prepares for the mid-April new year Songkran holiday.
"Do not panic because that is what this group wants," he said.
Prayut was speaking in Bangkok before accompanying Prime Minister Yingluck Shinawatra on a visit to the largest southern city of Hat Yai, where a car bomb at a hotel triggered a fire that killed three people and injured more than 400.
The attack came about an hour after car bombs killed 11 people and wounded more than 100 in the town of Yala further south.
The hotel attack was an apparent escalation in the tactics of militants who have waged a deadly insurgency in the far south since 2004 that has claimed both Buddhist and Muslim lives.
Hat Yai, a popular destination for tourists from Malaysia and Singapore, has largely been spared the violence which plagues the three neighbouring far southern provinces on an almost daily basis but rarely on the scale of Saturday's attacks.
The insurgents are not thought to be part of a global jihad movement but are instead rebelling against a long history of perceived discrimination against ethnic-Malay Muslims by successive Thai governments.
Struggling to quell the unrest, authorities have imposed emergency rule in the region, which rights campaigners say effectively gives the army legal immunity.
The region was an autonomous Malay Muslim sultanate until it was annexed in 1902 by mainly Buddhist Thailand.