When the battle of Chickamauga began, General Negley's division was on the move, marching to the sound of the artillery, and it reached the field just in time to push forward on the right and fill up a gap created by the dispersion of General Van Cleve's division. In the desperate fight which ensued, the rebel General Preston Smith was killed, and the enemy driven in confusion. On the second day of the battle General Negley's division was not so fortunate. One brigade was sent to the extreme left, another was placed in the centre, and the third held in reserve. Later in the day the general himself was taken from the command of the division and ordered to the command of a number of batteries which were concentrated on a hill on a new line to which it was proposed to retire, and which were intended to cover the retrograde movement. Before this manœuvre could be executed, however, the right wing and centre of the army were broken, and the troops fell back in confusion. The enemy charged upon the guns of General Negley in great force, and, moving upon the flanks, greatly threatened their capture. By great exertions the general succeeded in carrying them from the field without the aid of any infantry supports, and thus saved about fifty guns from capture.
On retiring to Rossville, he found himself, in the absence of Rosecrans, McCook, and Crittenden at Chattanooga, the senior officer in that part of the field, and he immediately began the work of reorganizing the troops of the several divisions gathered indiscriminately there. He succeeded in reorganizing a large number of men, and, selecting a strong position at Rossville Gap, endeavored to open communication with General Thomas. This was found impracticable, however. During the night General Thomas retired to this position, and, forming a junction with General Negley, ordered him to post the forces along the line selected by him, and prepared to give the enemy a warm reception on the next morning. Bragg was too wise to attack, and contented himself with merely reconnoitering the position. On the succeeding day the troops were retired to Chattanooga, and preparations were made for the siege which followed. During this siege General Negley was relieved from duty by General Rosecrans in such a manner and so unjustly that he was induced to demand an examination into his official conduct. This was granted; a court of inquiry was convened and an investigation made, resulting in General Negley's acquittal. The official record of the court states in conclusion "that General Negley exhibited throughout the day (the second day of the battle) and the following night great activity and zeal in the discharge of his duties, and the court do not find in the evidence before them any ground for censure." General Negley, on the conclusion of the trial, was ordered to report to the Adjutant General at Washington, and did so, but soon after resigned. He is now engaged in the cultivation of his farm near Pittsburg, Pennsylvania.