The single-chain variable fragment (scFv) of monoclonal antibodies is a promising recombinant nanostructure for various medical applications, including bioimaging and targeted therapy. While numerous scFv antibodies against eukaryotic cell surface proteins (especially cancer biomarkers) have been generated and engineered to suit various purposes, only a few specific scFv against bacterial cell surfaces have been developed, especially those of human origin. Recent incidents of emerging multidrug-resistant pathogenic bacteria and the realization of the importance of a balanced microbiota on the health of the host has led to more interests in the development of recombinant antibacterial antibodies as a detection probe or targeted therapy for bacterial infections. This study reports the generation of two specific human antibacterial scFv using phage display antibody technology. The recombinant scFv fragments of about 30 kDa and a diameter of 5 nm were produced and purified from engineered that can enhance cytosolic disulfide bond formation. As a proof of principle, and were used as model Gram-positive and Gram-negative bacteria, respectively. Specificity at the strain and species level to both planktonic and biofilm forms of these bacteria were demonstrated in various assay formats, namely, ELISA, flow cytometry, western blot, immunofluorescence, and electron microscopy via the hexa-histidine tag. This recombinant scFv generation platform can be applied for other bacteria, and since the scFv obtained has a benefit of being a human origin, it could be conveniently engineered for various therapeutic or theranostic applications with minimized adverse immunoreaction.