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Barley stem gall midge, Mayetiola hordei (Kieffer) (Diptera: Cecidomyiidae) is among the most destructive pests of barley (Hordeum vulgare L.) in the Mediterranean region of southern Europe and northern Africa. In Tunisia, the biology and ecology of this pest are not well documented. In addition to a management study, a study of M. hordei distribution, frequency of occurrence and population dynamics in barley crops was conducted in two semi-arid regions of North Tunisia. The pest was detected in 84% of all sampled fields across two years. Zaghouan region was identified as a high spot region with economic infestations registered in 67 and 56% of surveyed fields in 2013 and 2014, respectively. Three annual generations occurred at both sites, with two complete and one incomplete generation. The second generation (winter generation) is the most important one in terms of crop damage. The study showed that damage from this pest affects host plant growth at different developmental stages. The highest infestation was observed during tillering and elongation. The impact of different sowing date of barley on M. hordei infestation was also conducted. The infestation rate of barley by M. hordei increased by delaying sowing date. We demonstrate that M. hordei is a serious biosecurity threat to Tunisian barley’s production and industry.