The Delfin served in basic, intermediate and weapons training roles. For this latter mission, they were equipped with hardpoints to carry gunpods, bombs or rockets, and thus armed, Egyptian L-29s were sent into combat against Israeli tanks during the Yom Kippur War. The L-29 was supplanted in the inventory of many of its operators by the Aero L-39 Albatros. More than 2,000 L-29s were supplied to the Soviet Air Force, acquiring the NATO reporting name "Maya."
L-29's, along with the newer L-39,were used extensively in ground attack missions in the Nagorno-Karabakh War by Azeri forces. At least 14 were shot down by Armenian air-defences, out of the total inventory of 18 L-29's. The Azeri Air Force lost large amounts of its air force due to anti aircraft fire.
As a trainer, the L-29 enabled air forces to adopt an "all-through" training on jet aircraft, replacing earlier piston-engined types.
On July 16, 1975, a Czechoslovak Air Force L-29 shot down a Polish civilian biplane piloted by Dionizy Bielanski that was attempting to defect to the West.
On October 2, 2007, an unmodified L-29 was used for the world’s first jet flight powered solely by 100% biodiesel fuel. Pilots Carol Sugars and Douglas Rodante flew their Delphin Jet from Stead Airport, Reno, Nevada to Leesburg International Airport, Leesburg, Florida in order to promote environmentally friendly fuels in aviation.
From September 10 to September 14, 2008, two L-29s took first and second place at the Reno Air Races. Both L-29s consistently posted laps at or above 500 miles per hour. Former Astronaut Curt Brown took first place in "Viper," followed by Red Bull racer Mike Mangold in "Euroburner."
Russia says it destroyed two Georgian L-29s during the 2008 South Ossetia war.
The Separatists in the Ukrainian Civil War claimed to have an operational L-29 on the 18th of January 2015.