AbstractGemmata spp. bacteria thrive in the same aquatic environments as free-living amoebae. DNA-based detection of Gemmata spp. sequences in the microbiota of the human digestive tract and blood further questioned the susceptibility of Gemmata spp. to phagocytes. Here, Gemmata obscuriglobus and Gemmata massiliana were co-cultured with the amoebae Acanthamoeba polyphaga, Acanthamoeba castellanii, Acanthamoeba griffini and THP-1 macrophage-like phagocytes. All experiments were performed in five independant replicates. The ratio amoeba/bacteria was 1:20 and the ratio THP-1/bacteria was 1:10. After a 2-hour co-culture, extracellular bacteria were killed by kanamycin or amikacin and eliminated. The intracellular location of Gemmata bacteria was specified by confocal microscopy. Microscopic enumerations and culture-based enumerations of colony-forming units were performed at T = 0, 1, 2, 3, 4, 8, 16, 24, 48 and 72 hours post-infection. Then, Gemmata bacteria were engulfed into the phagocytes’ cytoplasmic vacuoles, more than (98 ± 2)% of Gemmata bacteria, compared to controls, were destroyed by phagocytic cells after a 48-h co-culture according to microscopy and culture results, and no positive culture was observed at T = 72-hours. Under our co-culture conditions, Gemmata bacteria were therefore susceptible to the environmental and host phagocytes here investigated. These data suggest that these Acanthamoeba species and THP-1 cells cannot be used to isolate G. massiliana and G. obscuriglobus under the co-culture conditions applied in this study. Although the THP-1 response can point towards potential responses that might occur in vivo, these responses should first bevalidated by in vivo studies to draw definite conclusions.