Human amniotic fluid cells are immune-privileged with low immunogenicity and anti-inflammatory properties. They are able to self-renew, are highly proliferative, and have a broad differentiation potential, making them amenable for cell-based therapies. Amniotic fluid (AF) is routinely obtained via amniocentesis and contains heterogeneous populations of foetal-derived progenitor cells including mesenchymal stem cells (MSCs). In this study, we isolated human MSCs from AF (AF-MSCs) obtained during Caesarean sections (C-sections) and characterized them. These AF-MSCs showed typical MSC characteristics such as morphology, in vitro differentiation potential, surface marker expression, and secreted factors. Besides vimentin and the stem cell marker CD133, subpopulations of AF-MSCs expressed pluripotency-associated markers such as SSEA4, c-Kit, TRA-1-60, and TRA-1-81. The secretome and related gene ontology (GO) terms underline their immune modulatory properties. Furthermore, transcriptome analyses revealed similarities with native foetal bone marrow-derived MSCs. Significant KEGG pathways as well as GO terms are mostly related to immune function, embryonic skeletal system, and TGFβ-signalling. An AF-MSC-enriched gene set included putative AF-MSC markers PSG5, EMX-2, and EVR-3. In essence, C-section-derived AF-MSCs can be routinely obtained and are amenable for personalized cell therapies and disease modelling.