Brucellosis is a widespread disease in Egypt which cause huge economic losses in the dairy industry. The present study aims at isolating and identifying Brucella (B.) spp. circulating in bovine and buffalo dairy herds kept at farmers houses in four districts of the Delta region of Egypt. One hundred and five tissue specimens were collected from seropositive cattle and buffaloes. The samples included 10 vaginal swabs, 3 placentas, 3 uteri and 86 supra-mammary lymph nodes from dams, as well as 3 stomach contents from aborted fetuses. Matrix-assisted laser desorption ionization (MALDI) and the conventional biotyping techniques were used for preliminary identification of isolates into the genus level. AMOS-PCR was applied to differentiate Brucella isolates into species level. Nineteen Brucella strains have been identified, four B. abortus strains were recovered from cattle and 15 B. melitensis strains were isolated from both cattle (n = 8) and buffaloes (n = 7). The predominant occurrence of B. melitensis in bovines raises the fact that B. melitensis clone can cross species barriers and can establish a permanent reservoir in cattle and buffaloes. Presence of culture-positive animals at householders represent a high-risk factor for human infection. This knowledge is of significant importance in the control of brucellosis in bovines.