The frequent detection of antibiotics in the environment is causing great concern in scientific communities and public health agencies due to the potential adverse health effects the chemicals may have on aquatic life, animals and possibly humans. The major concern associated with this contamination is related to the selection and development of resistance and its implications for human medicine. Ciprofloxacin (CIP), the focus of this research, is an antibiotic frequently detected in aquatic environments. It has been detected in surface waters from 9 ng/L up to 30 ng/L and in hospital effluents from 0.7 μg/L to 125 μg/L. Previous studies have shown that ozonation is an effective treatment method to remove antibiotics from water and wastewater. However, there is limited data on the potential development of resistance in microorganisms from exposure to reaction-intermediates formed during ozonation. The objective of this research was to investigate the possibility and rate of development of resistance in E. coli exposed to CIP solutions pre-oxidized at different levels. Analysis of the potential for the development of resistance was organized around the ozonation of 1000 μg/L CIP solution to various degrees of oxidation and exposing the pre-oxidized solution to initially non-resistant E. coli. Five oxidation levels at 98.03, 98.84, 99.22, 99.93, and 100% CIP removal were used. The equivalent concentrations of these five oxidation levels were 19.5, 11.7, 7.81, 0.7, and 0 μg/L residual CIP. Three non-oxidized CIP exposures were conducted at concentrations comparable to three pre-oxidized CIP at 19.5, 11.7, and 7.81 μg/L to compare the effects of non-oxidized CIP and pre-oxidized CIP exposures on initially non-resistant E. coli. The mean Minimum Inhibitory Concentrations (MIC) were determined for 12, 24, 48, and 72-hour exposures and compared to MIC of the control (0 μg/L) to evaluate the potential for the development of resistance in E. coli. The results showed that the pre-oxidized CIP resulted in higher MICs than the control at all exposure times. This may indicate the development of a novel resistance by mutation of the bacteria or an up-regulation of an existing dormant gene. Some pre-oxidized CIP and non-oxidized CIP exposures showed to be significantly different from the control. As the exposure time increased, the MICs of both the pre-oxidized and non-oxidized CIP solutions became significantly different from the control, indicating an increased potential for development of resistance. The results comparing the pre-oxidized CIP and the non-oxidized CIP solutions showed little to no significant difference for the 12, 24, and 72-hour exposures. However, the 48-hour exposure results showed a significant difference between the pre-oxidized CIP at 98.03% removal (19.5 μg/L residual CIP) and the non-oxidized CIP at 19.5 μg/L.