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Aims:  Isolation and antimicrobial evaluation of aquatic bacterial strains from two cenotes. Methods and Results:  A total of 258 bacterial strains were isolated from the water and sediment of two cenotes in the Yucatan peninsula, all of which were screened against six pathogenic micro-organisms. Antimicrobial activity was detected in 46 of the isolated strains against at least one of the target strains tested. Antimicrobially active isolates were identified as: Aeromonas, Bacillus, Burkholderia, Photobacterium, Pseudomonas, Serratia, Shewanella, Stenotrophomonas genera, and 13 remained unidentified. All antimicrobially active strains were able to grow in salt medium at a concentration of 75 g l−1, thus classifying as moderately halotolerant bacteria. Most of the antimicrobially active strains exhibited a broad action spectrum, where 61% was because of uncharacterized antimicrobial substances, 25% because of bacteriocins and 13% because of siderophores. Ten strains were able to biosynthesize biosurfactant metabolites. Conclusions:  Native bacteria from the Yucatan peninsula showed an interesting antimicrobial activity, diverse mode of action and moderate halotolerance to salt. Significance and Impact of the Study:  This is the first report on bacterial isolates from cenotes of the Yucatan peninsula and their antimicrobial characterization, with great potential for future biotechnological applications. [ABSTRACT FROM AUTHOR]