Ninety buffalo's milk samples included 20 bulk tank milk samples from small dairy farms and 70 from retail milk at different localities in Dakahlia Governorate. Forty-eight individual milk specimens were obtained from animals positive for Rose Bengal Test (RBT), buffered acidified plate antigen test (BAPAT) together. Milk samples were tested with a milk ring test (MRT), followed by isolation and identification of the prevalent serotypes. At the same time, conventional PCR was applied. One hundred and sixty seven buffalo's serum samples were collected from 12 smallholder's herds and their bulk tanks gave positive to MRT were confirmed by BAPAT and RBT. A total of 50 karish cheese were collected from street vendors and supermarkets located in Mansoura city, Dakahlia Governorate, Egypt. Conventional PCR was also used to detect Brucella DNA at the species level on the collected cheese samples. The results showed that the prevalence of brucellosis in small dairy buffalo farms was 60% by MRT and 28.7% though using RBT and BAPAT. The prevalence of Brucella in buffalo's retail milk was 20% in each of Bilqase, Sherbin, and Talkha, 30% in each of Aga and Nabroh, 10% in Sinblaween. However no positive results was revealed in Mansoura. The isolation rate of Brucella was 31.25% in individual positive samples and 7.69% in retail positive milk samples. While using conventional PCR, the percentage reached 62.5% in individual positive samples and 69.23% in retail positive milk samples. All Brucella isolates (n = 16) were biochemically identified into three Brucella abortus biovar1 (18.75%)and 13 Brucella melitensis biovar 3 (81.25%). The prevalence of Brucella in examined Karish cheese was 20%. All of the tested isolates were verified using conventional PCR. Raw milk and unpasteurized soft cheese have public health risks for humans and are possible sources of transmitting Brucella and must be under regular and mandatory food control measures.