Fragment antigen-binding domains of antibodies (F_abs) are powerful probes of structure-function relationships of assembly line polyketide synthases (PKSs). We report the discovery and characterization of F_abs interrogating the structure and function of the ketosynthase-acyltransferase (KS-AT) core of Module 2 of the 6-deoxyerythronolide B synthase (DEBS). Two F_abs (AC2 and BB1) were identified to potently inhibit the catalytic activity of Module 2. Both AC2 and BB1 were found to modulate ACP-mediated reactions catalyzed by this module, albeit by distinct mechanisms. AC2 primarily affects the rate (k_cat), whereas BB1 increases the K_M of an ACP-mediated reaction. A third F_ab, AA5, binds to the KS-AT fragment of DEBS Module 2 without altering either parameter; it is phenotypically reminiscent of a previously characterized F_ab, 1B2, shown to principally recognize the N-terminal helical docking domain of DEBS Module 3. Crystal structures of AA5 and 1B2 bound to the KS-AT fragment of Module 2 were solved to 2.70 and 2.65 Å resolution, respectively, and revealed entirely distinct recognition features of the two antibodies. The new tools and insights reported here pave the way toward advancing our understanding of the structure-function relationships of DEBS Module 2, arguably the most well-studied module of an assembly line PKS.