The stomach acts as a barrier to ingested microbes, thereby influencing the microbial ecology of the entire gastrointestinal (GI) tract. The stomach microbiota and the role of human host and environmental factors, such as health status or medications, in shaping its composition remain largely unknown. We sought to characterize the bacterial and fungal microbiota in the stomach fluid in order to gain insights into the role of the stomach in GI homeostasis. Gastric fluid was collected from 25 patients undergoing clinically indicated upper endoscopy. DNA isolates were used for PCR amplification of bacterial 16S ribosomal RNA (rRNA) genes and fungal internal transcribed spacers (ITS). RNA isolates were used for 16S rRNA cDNA generation and subsequent PCR amplification. While all stomach fluid samples are dominated by the phyla Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria and Fusobacteria (>99% of sequence reads), the transcriptionally active microbiota shows significant reduction in Actinobacteria (34%) and increase in Campylobacter (444%) (P<0.003), specifically the oral commensal and suspected intestinal pathogen Campylobacter concisus. Bacterial but not fungal diversity is reduced by antibiotic treatment (28%; P<0.02), immunosuppression in transplant recipients and HIV/AIDS patients (42%; P<0.001) and gastric fluid pH >4 (70%; P<0.05). Immunosuppression correlates with decreased abundance of Prevotella (24%), Fusobacterium (2%) and Leptotrichia (6%) and increased abundance of Lactobacillus (3844%) (P<0.003). We have generated the first in-depth characterization of the human gastric fluid microbiota, using bacterial 16S rRNA gene and transcript, and fungal ITS amplicon sequencing and provide evidence for a significant impact of the host immune status on its composition with likely consequences for human health.