Antibiotic residues have been detected in aquatic environments worldwide. Biofilms are one of the most successful life forms, and as a result are ubiquitous in natural waters. However, the response mechanism of freshwater biofilms to the stress of various antibiotic residues is still unclear. Here, the stress of veterinary antibiotic florfenicol (FF) and fluoroquinolone antibiotic ofloxacin (OFL) on freshwater biofilms were investigated by determining the changes in the key physicochemical and biological properties of the biofilms. The results showed that the chlorophyll a content in biofilms firstly decreased to 46–71% and then recovered to original content under the stress of FF and OFL with high, mid, and low concentrations. Meanwhile, the activities of antioxidant enzymes, including superoxide dismutase and catalase, increased between 1.3–6.7 times their initial values. FF was more toxic to the biofilms than OFL. The distribution coefficients of FF and OFL binding in extracellular polymeric substances (EPS)-free biofilms were 3.2 and 6.5 times higher than those in intact biofilms, respectively. It indicated that EPS could inhibit the FF and OFL accumulation in biofilm cells. The present study shows that the EPS matrix, as the house of freshwater biofilms, is the primary barrier that resists the stress from antibiotic residues.