Brucellosis is a relatively common zoonosis, particularly in the developing countries. Our aim was to cultivate the Brucella wild-type strain from eggs of Dermacentor marginatus, and to molecularly investigate the presence of Brucella DNA in different developmental stages. A total of 350 engorged adult female ticks were collected from sheep and cattle, and individually placed into a single clean breathable tube suitable for laying eggs and for the development of larvae. Detection of Brucella DNA was attempted in engorged female ticks and their offsprings based on the Brucella outer membrane protein gene 22 (omp22) and IS711 gene. Additionally, bacterial isolation in the culture medium of Brucella was performed. The eggs from the Brucella DNA-positive engorged female ticks were positive for Brucella omp22 (4.6%, 16/350). The prevalence was 40.9% (90/220) in larvae, which developed from the Brucella DNA-positive egg batch. Based on BLASTn analysis of IS711 gene, ten (4.1%, 10/242) PCR products were identified as Brucella melitensis in D. marginatus from sheep, while six (5.6%, 6/108) were identified as B. abortus in D. marginatus infesting cattle. B. melitensis biotype 3 was isolated from eggs of D. marginatus. This is the first study which demonstrates the transovarial transmission of Brucella in the D. marginatus. Furthermore, Brucella may be partially transmitted from D. marginatus eggs to larvae (40.9%) in D. marginatus. These findings extend our knowledge on the transmission of Brucella in D. marginatus offsprings.