In the lead-up to the First Balkan War, the regiment was mobilized on 17 September 1912, again as part of the 2nd Division, in the Army of Thessaly. With the outbreak of the war on 5 May, it crossed the border and participated in the opening skirmishes of the Battle of Elassona. On 9 October it fought in the Battle of Sarantaporos in the area of Vigla. Following the Greek victory there, the regiment advanced in the direction of Kozani. During the Battle of Giannitsa on 18 October, the regiment was detached as reinforcements to the 3rd Infantry Division. Following the capture of Thessaloniki, in November the 3rd Battalion furnished troops for the Greek fleet's landing operations against various Ottoman-held Aegean islands, notably participating in the capture of Chios. After that, the 3rd Battalion was moved by sea to the Epirus front, followed by the 1st and 2nd Battalions that moved over land. The latter participated in operations in the directions of Delviniko, but by February 1913 the entire regiment was gathered, as part of the 2nd Division, before the Ottoman fortified position of Bizani, that covered the city of Ioannina. The regiment held the sector of the Avgo height, at the centre of the Greek front for the Battle of Bizani. After the capture of Ioannina, the regiment participated in the Greek army's northward advance in the direction of Delvinë–Sarandë–Gjirokaster. In March 1913, it returned to Thessaloniki.