Microbial pathogen entry and survival in the host is mediated by a network of molecular interactions between the two partners, which has been the subject of many research efforts. A complex picture is emerging in which host‐pathogen crosstalk involves a high number of proteins, often with redundant functions. In the present study, we investigated the potential of protein microarrays to simultaneously scan interactions between surface proteins from two main human streptococcal pathogens, Streptococcus pyogenes and Streptococcus agalactiae, and three human ligands, fibronectin, fibrinogen, and C4 binding protein, known to play an important role in streptococcal pathogenesis. By using this technology, we confirmed interactions described in the literature and detected a novel set of streptococcal proteins with binding capacities for the human ligands. The observations were validated by Western blot and ELISA techniques. Three of the newly identified proteins were isoforms of a group B streptococcus‐secreted component named Fib and displayed differential binding capacities for fibronectin, fibrinogen, and C4BP. The protein regions involved in the interaction with each ligand were identified by constructing fragments of one of the Fib variants. The approach proved valuable for the acquisition of novel insights into the complex network of protein‐protein interactions occurring during microbial infection.—Margarit, I., Bonacci, S., Pietrocola, G., Rindi, S., Ghezzo, C., Bombaci, M., Nardi‐Dei, V., Grifantini, R., Speziale, P., Grandi, G. Capturing host‐pathogen interactions by protein microarrays: identification of novel streptococcal proteins binding to human fibronectin, fibrinogen and C4BP. FASEBJ. 23, 3100‐3112 (2009). www.fasebj.org