Most protocols regarding sample decontamination for Mycobacterium avium subspecies paratuberculosis (MAP) isolation are based on the MAP detection from feces and not milk. The choice of the best decontamination protocol is crucial to a successful MAP isolation. In this study, 36 combinations of variables for sample decontamination and MAP isolation from raw milk presented in the literature were carried out on milk samples artificially contaminated which were then inoculated into tubes with three different culture media: Herrold egg yolk medium (HEYM) prepared with fresh egg yolk, HEYM prepared with commercial egg yolk and Lowenstein-Jensen medium (LJ). Each treatment was performed in triplicate for each medium, with a total of 324 observations. The protocol combination which provided higher MAP growth and lower nonspecific contamination in a shorter period of time was considered improved. In this study, the protocol involving 0.75% HPC at room temperature for 24 h, using centrifuge at 2500 × g for 15 min and addition of antimicrobial solution immediately before inoculation into tubes with HEYM prepared with fresh egg yolk provided the greatest MAP isolation from raw milk samples.