Routine serological diagnosis of toxoplasmosis provides high sensitivity, but specificity varies depending on the test used; false-positive results (IgM) have been reported. Blood samples were collected from 88 women (59 pregnant and 29 nonpregnant) and 86 contact animals (62 sheep and 24 goats) at El Fayoum Governorate during the period from October 2005 to December 2006. All collected samples were tested for Toxoplasma gondii infection by serological tests (ELISA IgM & IgG and Sabin-Feldman dye test) and polymerase chain reaction (PCR). Results revealed specific IgG in 45.8% and 41.4%, IgM in 30.5% and 24.2%, and positive Sabin-Feldman dye test in 23.7% and 17.2% in pregnant and nonpregnant women, respectively. Positive PCR products were detected in 32.2% and 27.6% in pregnant and nonpregnant women, respectively. Regarding animals, positive ELISA IgG and PCR were detected in 98.4% and 67.7% of sheep and 41.7% and 25.0% of goats, respectively. It was concluded that serological tests can detect higher rate of toxoplasmosis than PCR, so ELISA combined with the PCR technique is a recommended tool for accurate diagnosis of toxoplasmosis.