Medicine Sans Frontiers or MSF treated 116 people with gunshot wounds following heavy fighting in Malakal and Nassir of Upper Nile state.
In a statement issued on Wednesday, MSF said heavy fighting in Upper Nile, Unity and Jonglei states left thousands of people newly displaced and hundreds wounded.
MSF Head of Mission in South Sudan Raphael Gorgeu said the fighting limited their ability to reach displaced people in refuge.
He added that the fighting was preventing victims from receiving medical and humanitarian assistance.
Mr Gorgeu said they continue treating more wounded patients in hospitals.
He expressed concerns with the conditions of hundreds of thousands of displaced people across the country with little food, water or access to healthcare.
MSF called on the warring parties to respect the integrity of medical facilities, allow aid organisations to access affected communities and allow patients to receive medical treatment.