Environmental water sources are constantly polluted by anthropogenic compounds, not always minimized by conventional water treatment methods to remove these compounds at the micro- and nano-range. The absolute concentrations of a suite of seven representative environmental micropollutants were compared pre- and post-treatment with both ozone and microbial biofilms, in terms of removal efficiencies and toxicity assays. Both synthetic micropollutant mixes and environmental water samples were evaluated. The study started with two representative micropollutants (carbamazepine, CBZ, and sulfamethoxazole, SMX), and broadened into a suite of pollutants, evaluating whole-sample eco-toxicological footprints. An ozone concentration of 4.24 ± 0.27 mg/L in tap water, resulted in an 87.9% and 96.5% removal of CBZ and SMX, respectively, within 1 min. Despite almost immediate removal of parent micropollutants by oxidation, endocrine disruption potential (anti-estrogenicity) of CBZ and SMX required up to 240 min of ozone treatment to show no assay effect. A broader suite of micropollutants in more complex environmental matrices showed scavenging of ozone (2.95 ± 0.17–0.25 ± 0.03 mg/L) and varying micropollutant recalcitrance to oxidation. Lower matrix pollution led to lower reduction in eco-toxicity. Microbial degradation of CBZ and SMX (56% and 70% versus 19% and 79%, respectively, in duplicate biofilms) by nutrient-limited biofilms showed less removal than ozonation, with marked variation due to the stochastic nature of biofilm sloughing. Microbial degradation of CBZ and SMX resulted in an increase of >90% in both estrogenicity and Aliivibrio inhibition. The results obtained from this study address a gap in understanding the removal efficiency of micropollutants, where the removal process often receives more attention than the comparative reduction of toxicological effects. This shift from a controlled laboratory environment to real-world scenarios also provided comparative insights into the removal of micropollutants and the eco-toxicity of the transformation by-products of each process.