Navicula sp., a type of benthic diatom, plays a crucial role in the carbon cycle as a widely distributed algae in water bodies, making it an essential primary producer in the context of global carbon neutrality. However, using erythromycin (ERY) and levofloxacin (LEV) in medicine, livestock, and aquaculture has introduced a new class of pollutants known as antibiotic pollutants, which pose potential threats to human and animal health. This study aimed to investigate the toxic effects of ERY and LEV, individually or in combination, on the growth, antioxidant system, chlorophyll synthesis, and various cell osmotic pressure indexes (such as soluble protein, proline, and betaine) of Navicula sp. The results indicated that ERY (1 mg/L), LEV (320 mg/L), and their combined effects could inhibit the growth of Navicula sp. Interestingly, the combination of these two drugs exhibited a time-dependent effect on the chlorophyll synthesis of Navicula sp., with ERY inhibiting the process while LEV promoted it. Furthermore, after 96 h of exposure to the drugs, the activities of GSH-Px, POD, CAT, and the contents of MDA, proline, and betaine increased. Conversely, the actions of GST and the contents of GSH and soluble protein decreased in the ERY group. In the LEV group, the activities of POD and CAT and the contents of GSH, MDA, proline, and betaine increased, while the contents of soluble protein decreased. Conversely, the mixed group exhibited increased POD activity and contents of GSH, MDA, proline, betaine, and soluble protein. These findings suggest that antibiotics found in pharmaceutical and personal care products (PPCPs) can harm primary marine benthic eukaryotes. The findings from the research on the possible hazards linked to antibiotic medications in aquatic ecosystems offer valuable knowledge for ensuring the safe application of these drugs in environmental contexts.