Antibiotics are frequently applied to treat bacterial infections in humans and animals. However, most consumed antibiotics are excreted into wastewater as metabolites or in their original form. Therefore, removal of antibiotics from aquatic environments is of high research interest. In this study, we investigated the removal of sulfamethoxazole (SMX) and ofloxacin (OFX) with Chaetoceros muelleri, a marine diatom. The optimization process was conducted using response surface methodology (RSM) with two independent parameters, i.e., the initial concentration of antibiotics and contact time. The optimum removal of SMX and OFX were 39.8% (0.19 mg L−1) and 42.5% (0.21 mg L−1) at the initial concentration (0.5 mg L−1) and contact time (6.3 days). Apart from that, the toxicity effect of antibiotics on the diatom was monitored in different SMX and OFX concentrations (0 to 50 mg L−1). The protein (mg L−1) and carotenoid (μg L−1) content increased when the antibiotic concentration increased up to 20 mg L−1, while cell viability was not significantly affected up to 20 mg L−1 of antibiotic concentration. Protein content, carotenoid, and cell viability decreased during high antibiotic concentrations (more than 20 to 30 mg L−1). This study revealed that the use of Chaetoceros muelleri is an appealing solution to remove certain antibiotics from wastewater.