Acute hepatopancreatic necrosis disease (AHPND) outbreaks caused by Vibrio parahaemolyticus (VPAHPND) emerged in Mexico in 2013, causing a 65% drop in shrimp tonnage produced. This work aimed to isolate bacterial strains from Mexican marine ecosystems antagonistic towards VPAHPND. Among 258 bacterial isolates, only 17 showed antagonist activity in vitro, but only (Bacillus pumilus‐Y100, B. pumilus‐Y119, B. pumilus‐43, Vibrio alginolyticus‐32a and V. campbellii‐H‐A) were chosen for bioassay challenge. These probiotic bacteria were sprayed on commercial shrimp pellets, and experimental infection was carried out by immersing the shrimp in VPAHPND inoculum. Bacterial strains 32a, Y100 and H‐A significantly reduced the cumulative mortality of shrimp exposed to AHPND (4.76%, 12.54% and 16% respectively). Y119 and 43 did not show any beneficial effect against VPAHPND but were pathogenic in previous assays with post‐larval white shrimp and bioassay trials. Concerning the qPCR results, the presence of low numbers of DNA copies of VPAHPND was found only in the H‐A trial, suggesting that H‐A exerts a bacteriostatic effect; in contrast, the results for 32a, Y100, Y119 and 43 were negative, indicating that these strains exhibited a microbicidal effect against VPAHPND. The histopathological results agreed with the qPCR results and did not show compatible lesions caused by AHPND. In conclusion, 32a and Y100 seem to be the first antagonists to specifically inhibit VPAHPND in vivo.