Fidaxomicin (FDX) is a marketed antibiotic for the treatment Clostridium difficile infections (CDI). Although showing interesting antibacterial properties against many Gram-positive bacteria, the application of this antibiotic is currently limited to treatment of CDI. Semisynthetic modifications present a promising strategy to improve its pharmacokinetic properties and also circumvent resistance development by broadening the structural diversity of derivatives. Based on a rational design using a cryo-EM structure analysis, we implemented two strategic site- selective catalytic reactions with a special emphasis to study the role of the carbohydrate units. Site-selective introduction of various ester moieties on the noviose as well as a Tsuji-Trost type rhamnose cleavage allowed the synthesis of novel fidaxomicin analogs with promising antibacterial activities against C. difficile and M. tuberculosis.