The identification of single-chain antibodies (scFvs) that interfere in vivo with the building of the complex that activate the prokaryotic, sigma54-dependent promoter Pu of the catabolic TOL plasmid pWW0 is reported. To this end, a phage M13 library of scFvs was raised against the cognate prokaryotic enhancer-binding activator, XylR. The scFv pool was then expressed intracellularly in a reporter Pu-lacZ strain of Escherichia coli designed to permit formation of intramolecular disulphide bonds in cytoplasmic proteins. This strain allowed the assembly of functional scFvs and the direct testing of their activity on the Pu promoter in vivo. Specifically, genetic screening for lacZ-minus colonies yielded a number of scFvs able to downregulate transcriptional output in live cells. Two antibody clones were purified and shown to inhibit the activity of the same promoter in vitro as well. These scFvs targeted the DNA-binding domain of XylR and its ATP binding site respectively. This work provides a proof of principle that mimetic regulatory factors can be derived from an antibody repertoire that specifically interact with given transcriptional activators. As assembly of initiation complexes is stimulated or inhibited by regulatory proteins we argue that anti-XylR scFvs operate as bona fide transcriptional inhibitors of the Pu promoter.