Virginiamycin is an antibiotic produced by Streptomyces virginiae. The recovery process involves a crystallization step (solvent evaporation and precipitation using an antisolvent). To systematically evaluate the industrial crystallization process, the finished product was characterized by granulometry, X-ray diffraction, optical microscopy, scanning electron microscopy, hot-stage microscopy, thermogravimetry, near-infrared spectroscopy, and microbiological activity. The industrial process was simulated on a laboratory scale, analyzing the particle chord length size distribution as a function of time and studying the crystal formation in real time using focused beam reflectance. Analysis of the industrial process through quality by design and design of experiments showed that the crystallization process also influenced the values of the microbiological activity, and the current operation did not result in optimal values. The industrial process was modified within the certified conditions, which resulted in eliminating unexpected stops due to crystallization fouling and yielded high microbiological activity values in the finished product.