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In an aquaponic system, fish and plants are cultivated together in a symbiotic environment where they mutually benefit, using significantly less water than traditional farming methods. The main aim of this study was to investigate the occurrence of antimicrobial resistance in two aquaponic systems implemented in two Chilean high schools using rainbow trout and lettuce cultures. When water samples (fish tank, biofilter, and plant raft) were analyzed over a three-month period, no resistance to oxytetracycline was detected, whereas the occurrence of resistance to florfenicol was rather small, ranging from 0.01% to 3.1% of bacterial culturable counts. Eighteen isolates were recovered from various sources as representatives of the florfenicol-resistant population, and all of them belonged to the Pseudomonas genus, showing a multi-drug-resistance phenotype and exhibiting simultaneous resistance to 7–13 antimicrobials. All isolates exhibited resistance to amoxicillin, chloramphenicol, florfenicol, and furazolidone and susceptibility to meropenem, oxytetracycline, oxolinic acid, flumequine, ciprofloxacin, and enrofloxacin. Five and two isolates carried the amphenicol-resistance-encoding genes floR and cmlA, respectively, whereas no carriage of integrons or the fexA, fexB, pexA, optrA, and cfr genes encoding for florfenicol resistance was detected. Eleven isolates carried plasmids, but only two of them were able to transfer their plasmid content by conjugation. The knowledge of the microbiome associated with aquaponic systems is still scarce, and their role as potential reservoirs of antimicrobial-resistant bacteria and related genes of these systems must be elucidated. [ABSTRACT FROM AUTHOR]