In this study, we re-assessed myxovalargin for its activity against M. tuberculosis and other pathogens and found the compound to exhibit a very low antitubercular (MIC of 0.2 µg/mL) and bactericidal activity, thus, opening a potential therapeutic window for application. We identified the biosynthetic gene cluster and biosynthetic reasoning followed by feeding experiments revealed that myxovalargins differ from the originally proposed structure, which was eventually corroborated by establishing a concise total synthesis of Myx. Next, the binding site of MyxA was visualized on the large ribosomal subunit using cryo-EM at between 2.1-2.5 Å resolution. The resolution enables a detailed description of the contacts that MyxA establishes with the ribosome, including water-mediated interactions, and provides a structural basis for the resistance mutations observed in M. tuberculosis. MyxA displayed good pharmacokinetic properties and showed no adverse effects after daily dosing of 2 mg/kg over 7 days. We demonstrated in vivo efficacy in a mouse model of Pasteurella multocida sepsis and then assessed myxovalargin in the more challenging mouse model of acute M. tuberculosis infection. Here, toxic effects after one week of treatment were observed highlighting the necessity of further improving the safety margin of Myx. Understanding the SAR is now feasible and the cryo-EM structure opens avenues for structure-based lead optimization of myxovalargin by using chemical and biotechnological production of analogues as a means to differentiate the specific inhibition of the ribosome from the unspecific toxic effect at higher doses.