Toxoplasma gondii is a protozoon parasite that can cause severe clinical problems such as congenital toxoplasmosis. The distribution of T. gondii genotypes varies from one geographic area to another. So far, little is known about the parasite genotypes in Tunisia, North Africa. The present study aimed isolating and genotyping T. gondii from the amniotic fluid (AF) and placenta of pregnant women in Monastir, Tunisia. Amniotic fluid and/or placenta from 80 women who acquired toxoplasma infection during pregnancy were tested by PCR and/or mouse bioassay. Genotyping of T. gondii isolates from these samples was performed with 15 microsatellite markers. Four viable T. gondii strains were isolated from either the AF or placenta of four women. Specifically, strains TUN001-MON1 and TUN002-MON2 were isolated from both the AF and placenta, TUN003-AHA from only the placenta, and TUN004-NEL from only the AF. The four viable strains were not virulent for mice. Genotyping revealed that the four strains were type II strains. This is the first report on isolation and genotyping of T. gondii from AF human samples in Tunisia. Further studies focused on T. gondii genotyping on a larger number of human cases and on animals in Tunisia are needed to improve the knowledge and epidemiology of toxoplasmosis.