Bovine brucellosis is a widespread zoonosis caused by Brucella abortus. The disease is prevalent nationwide in Iran and is on an increasing trend among humans and livestock. The eradication of brucellosis is challenging and requires control policies at both national and regional levels. Regarding this, the aim of the current study was to evaluate if Brucella is implicated in an abortion outbreak that occurred in a dairy cattle herd, in Shahre Rey, Tehran province, Iran, after vaccination with B. abortus Iriba vaccine. The research context was a dairy cattle farm with 2,000 animals located in Shahre Rey. This farm was Brucella-free based on the results of two serological tests performed one month before vaccination. After the incidence of the first case of abortion following vaccination, serodiagnosis revealed a seropositive reaction in 30 non-pregnant cows and 19 pregnant cows that aborted later. Bacteriology and molecular typing facilitated the identification of 16 isolates of B. abortus biovar 3 from the aborted animals. None of the isolates were confirmed as B. abortus Iribavaccine strain. The results confirmed that B. abortus biovar 3 was the most prevalent biovar in the cattle of Iran. The source and time of infection in the current study were not detected most likely due to the low biosecurity level in the farm (e.g., uncontrolled introduction of the agents via humans, infected animals, semen, and vectors). In endemic countries, the serodiagnosis of brucellosis alone is not sufficient and has to be accompanied by isolation and molecular diagnosis. In addition, it is important to evaluate the presence of B. abortus in bovine semen and vectors.