In the last decade, several phleboviruses transmitted by sand flies were detected in the Mediterranean countries, with the health impact of some of them being unknown. From September to October 2020, a total of 3351 sand flies were captured in Kherrata (Bejaia, northern Algeria) and identified by sex, grouped in 62 pools, which were tested for the presence of phlebovirus RNA using endpoint RT-PCR. Two pools (male and female, respectively) were positive. The genome sequencing and phylogenetic analysis showed that the two phleboviruses detected were closely related to the Punique virus (PUNV) isolated in Tunisia and detected in Algeria. Both PUNV strains were isolated on VERO cells from positive pools. Morphological identification of 300 sand flies randomly selected, showed a clear dominance of Phlebotomus perniciosus (98.67%). The dominance of this species in the study area was confirmed by PCR targeting the mitochondrial DNA. Our result represents the first isolation of PUNV and the second report in Algeria from two distinct regions which confirm its large circulation in the country and more broadly in North Africa. Further studies are needed to measure the impact on public health through seroprevalence studies in humans as well as animals and to investigate its potential involvement in neurological viral diseases.