Brucellosis is considered an economically important highly contagious and zoonotic bacterial disease of water buffaloes. Control of brucellosis in buffaloes is very important for public health. The efficacy of control program depends on the detection and eradication of infected animals coupled with vaccination and application of biosecurity. This study was carried out to control the brucellosis in buffalo farm in Assuit Governorate, Egypt during the period from April 2015 to August 2016. Out of 620 unvaccinated buffaloes, 87 (14.03%) aborted. Moreover, 90/620(14.51%), 82/620(13.22%), 82/620(13.22%), and 80/620 (12.9%) buffaloes were serologically positive by BAPA, RBPT, m SAT and Riv.T, respectively. Three isolates were differentiated as Brucella melitensis, biovar 3, one strain isolated from one vaginal swap out of 10 Riv.T. positive recently aborted buffaloes (10%) and two strains were isolated out of ten milk samples of Riv.T. positive buffaloes (20%). Eighty serological positive buffaloes to Riv.T were culled from the herd, while 60 serological negative heifers were vaccinated by Brucella abortus S 19 vaccine, with a dose of 3-8×109 cfu/5ml and monitored for serological titer for 240 days. After 6 months of vaccination, the number of serologically positive calves declined marginally to 50 (83.33%), 40 (66.67%), 50 (83.33%), 0 (0%), 40 (66.67%) and 0 (0%) by BAPA, RBPT, mSAT, CFT, iELISA and cELISA, respectively. Three successive serological tests every three weeks were done by screening tests, BAPA and RBPT and confirmed by Riv.T. At the end of the control program, all examined buffaloes were serologically negative. Application of biosecurity in the farm was applied by the sanitary disposal of aborted material and application of proper disinfectants at its recommended work strength and contact time.