Antibiotics used during production of food crops to control plant diseases may result in selection of antibiotic-resistant bacteria and occurrence of antibiotic residues. The aim of this research was to evaluate the effect of antibiotics used in butterhead lettuce production on persistence of commensal microbiota. Butterhead lettuce were treated with antibiotics (oxytetracycline, gentamicin, and streptomycin) at different concentrations (100, 200, 300, 400 and 500 ppm) starting at 5 weeks' growth by spraying once daily for 4 weeks and harvesting 7 days after the final spray application. The population of total aerobic bacteria and antibiotic-resistant bacteria were determined. The results showed antibiotic usage significantly decreased bacterial populations on lettuce. Moreover, increased concentration of antibiotics resulted in significantly greater decrease in bacterial populations. At a concentration of 500 ppm, all antibiotics achieved an approximate 2 log CFU/g decrease in bacterial populations. A stable population (4 log CFU/g) of potentially antibiotic-resistant commensal microbiota were maintained throughout production. Screening for level of susceptibility indicated that bacteria exhibited greater resistance to oxytetracycline than gentamicin. In conclusion, application of antibiotics failed to eliminate commensal microbiota, demonstrating large populations of antibiotic-resistant bacteria reside on lettuce grown under conditions used in the present study. This is the first study focused on antibiotic usage on hydroponic systems. Results of this study suggest regulations directed at antibiotic use on food crops must be developed and implemented to control the selection and spread of antibiotic-resistant bacteria that present a global health concern.