The current study deals with the Barn Owl (Tyto alba) diet from two Saharan localities in Ouargla, southeastern Algeria. This investigation was based on the owl regurgitated pellets contents, coupling the classical techniques, which consists of visual identification of prey specimens, and the environmental DNA metabarcoding method, applied to identify mammals and insects items. A total of 234 pellets with 456 specimens were analyzed. The identified preys belong to four classes, eight orders, 15 families and 26 species. Birds largely dominated (72.9% ≤ RA ≤ 89.6%) and the highest abundances were recorded by Streptopelia sp. (37.1 % ≤ RA ≤ 48.1%) and passer sp. (29.1% ≤ RA ≤ 33.02%). They are followed by rodents (9.4% ≤ RA ≤ 20.9%), mainly represented by Mus musculus (3.8 % ≤ RA ≤ 10.6%). Reptiles came in the third position (RA = 4.6%): among the consumed species were Gekkonidae sp. (RA= 3.7%) and Lacertidae sp. (RA = 0.8%). Insects constituted a negligible portion of the barn owl diet (0.9% ≤ RA ≤ 1.7%). The most consumed insects for each locality were Gryllotalpa sp. (RA = 0.85%) and Brachytrupes megacephalus (RA = 0.28%). The evenness index (E = 0.6) revealed that Tyto alba adopts a generalist and flexible feeding strategy. The DNA-based method allowed identifying seven prey species (one mammalian and six insects), out of which five (Pimelia sp., Lilioceris sp., Carpophilus sp., Attagenus pellio and Calliphora vicina) have not been confirmed by the classical method. Brachytrupes megacephalus and Mus musculus were identified by both techniques. Our findings provide baseline knowledge on bird diet species, using two complementary approaches. This constitutes vital elements to support ongoing studies on species management and conservation

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