This study evaluated the occurrence and potential proliferation of antibiotic resistance during biological wastewater treatment as a function of solids retention time (SRT), influent antibiotic concentrations, and temperature. Two phases of experiments were performed in laboratory-scale sequencing batch reactors (SBRs) fed with primary effluent from a full-scale wastewater treatment plant. Phase 1 evaluated SRTs of 2, 7, and 20 days with ambient antibiotic concentrations, and phase 2 evaluated a constant SRT of 7 days with influent antibiotic concentrations of 1×, 10×, and 100× relative to ambient levels. Ampicillin, sulfamethoxazole/trimethoprim, tetracycline, and vancomycin resistance were evaluated among Gram positive cocci (Staphylococcus and Streptococcus) using spread plate and minimum inhibitory concentration (MIC) assays. The laboratory-scale data demonstrated that biological treatment, in addition to longer SRTs, higher influent antibiotic concentrations, and higher temperatures, often resulted in greater relative prevalence of antibiotic resistance (up to 35% of the target population), and antibiotic resistant isolates were generally resistant to antibiotic concentrations 32 times higher than their baseline MICs. Some of these relationships were antibiotic-specific, with SRT having no significant impact on tetracycline resistance, influent antibiotic concentration having no significant impact on sulfamethoxazole/trimethoprim resistance, and temperature having no significant impact on vancomycin resistance.