Antibiotic cocktails are frequently used as secondary decontaminants prior to the culture of Mycobacterium avium subsp. paratuberculosis (MAP). This study investigated whether secondary incubation with an antibiotic cocktail containing vancomycin, nalidixic acid, and amphotericin B after primary exposure to N-acetyl-L-cysteine-1.5% sodium hydroxide affected the recovery of viable MAP from milkexperimentally spiked with 10² to 10⁶ cfu/ml. Results indicated that incubation with this antibiotic cocktail did decrease the incidence of contamination in culture media but it was also highly detrimental to the recovery of viableMAP. This effect was not advantageous given the low numbers of MAP naturally shed into milk and colostrum of infected cows. These results demonstrate that secondary incubation with antibiotics during the decontamination procedure could potentially lead to false-negative culture results because of their detrimental effect on the viability of MAP.