Background: Interest for the study of gut mycobiota in relation with human health and immune homeostasis has increased in the last years. From this perspective, new tools to study the immune/fungal interface are warranted. Systemic humoral immune responses could reflect the dynamic relationships between gut mycobiota and immunity. Using a novel flow cytometry technology (Fungi-Flow) to determine immunoglobulin (Ig) responses to fungi, we studied the relationships between gut mycobiota and systemic humoral anti-commensal immunity. Results: The Fungi-Flow method allows a sensitive and specific measurement of systemic IgG responses against 17 commensal and environmental fungi from the two main divisions; Ascomycota and Basidiomycota. IgG responses exhibited a high inter-individual variability. Anti-commensal IgG responses were contrasted with the relative abundance, alpha-diversity, and intra-genus richness of fungal species in gut mycobiota of twenty healthy donors. Categorization of gut mycobiota composition revealed two differentiated fungal ecosystems. Significant difference of anti-Saccharomyces systemic IgG responses were observed in healthy donors stratified according to the fungal ecosystem colonizing their gut. A positive and significant correlation was observed between the variety of IgG responses against fungal commensals and intestinal alpha-diversity. At the level of intra-genus species richness, intense IgG responses were associated with a low intra-genus richness for known pathobionts, but not commensals. Conclusions: Fungi-Flow allows an easy and reliable measure of personalized humoral responses against commensal fungi. Combining sequencing technology with our novel Fungi-Flow immunological method, we propose that there are at least two defined ecosystems in the human gut mycobiome associated with systemic humoral responses. Fungi-Flow opens new opportunities to improve our knowledge about the impact of mycobiota in humoral anti-commensal immunity and homeostasis.